diff --git a/modules/nf-core/sratools/prefetch/environment.yml b/modules/nf-core/sratools/prefetch/environment.yml
index cfc7d9a8363..0abad336cbc 100644
--- a/modules/nf-core/sratools/prefetch/environment.yml
+++ b/modules/nf-core/sratools/prefetch/environment.yml
@@ -4,4 +4,5 @@ channels:
   - bioconda
   - defaults
 dependencies:
-  - bioconda::sra-tools=3.0.8
+  - bioconda::sra-tools=3.1.0
+  - conda-forge::curl=8.5.0
diff --git a/modules/nf-core/sratools/prefetch/main.nf b/modules/nf-core/sratools/prefetch/main.nf
index 3c30739ab39..170f1753fba 100644
--- a/modules/nf-core/sratools/prefetch/main.nf
+++ b/modules/nf-core/sratools/prefetch/main.nf
@@ -4,8 +4,8 @@ process SRATOOLS_PREFETCH {
 
     conda "${moduleDir}/environment.yml"
     container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ?
-        'https://depot.galaxyproject.org/singularity/sra-tools:3.0.8--h9f5acd7_0' :
-        'biocontainers/sra-tools:3.0.8--h9f5acd7_0' }"
+        'https://depot.galaxyproject.org/singularity/sra-tools:3.1.0--h9f5acd7_0' :
+        'biocontainers/sra-tools:3.1.0--h9f5acd7_0' }"
 
     input:
     tuple val(meta), val(id)
diff --git a/modules/nf-core/sratools/prefetch/templates/retry_with_backoff.sh b/modules/nf-core/sratools/prefetch/templates/retry_with_backoff.sh
index a72a4bfb611..bfee60707a9 100755
--- a/modules/nf-core/sratools/prefetch/templates/retry_with_backoff.sh
+++ b/modules/nf-core/sratools/prefetch/templates/retry_with_backoff.sh
@@ -47,9 +47,19 @@ retry_with_backoff !{args2} \
     !{args} \
     !{id}
 
-[ -f !{id}.sralite ] && vdb-validate !{id}.sralite || vdb-validate !{id}
+# check file integrity using vdb-validate or (when archive contains no checksums) md5sum
+vdb-validate !{id} > vdb-validate_result.txt 2>&1 || exit 1
+if grep -q "checksums missing" vdb-validate_result.txt; then
+    VALID_MD5SUMS=$(curl --silent --fail --location --retry 3 --retry-delay 60 'https://locate.ncbi.nlm.nih.gov/sdl/2/retrieve?filetype=run&acc=!{id}')
+    LOCAL_MD5SUMS=$(md5sum !{id}/* | cut -f1 -d' ')
+    if ! grep -q -F -f <(echo "$LOCAL_MD5SUMS") <(echo "$VALID_MD5SUMS"); then
+        echo "MD5 sum check failed" 1>&2
+        exit 1
+    fi
+fi
 
 cat <<-END_VERSIONS > versions.yml
 "!{task.process}":
     sratools: $(prefetch --version 2>&1 | grep -Eo '[0-9.]+')
+    curl: $(curl --version | head -n 1 | sed 's/^curl //; s/ .*$//')
 END_VERSIONS
diff --git a/modules/nf-core/sratools/prefetch/tests/main.nf.test.snap b/modules/nf-core/sratools/prefetch/tests/main.nf.test.snap
index 946fc869ca1..67795ebbcd7 100644
--- a/modules/nf-core/sratools/prefetch/tests/main.nf.test.snap
+++ b/modules/nf-core/sratools/prefetch/tests/main.nf.test.snap
@@ -14,7 +14,7 @@
                     ]
                 ],
                 "1": [
-                    "versions.yml:md5,c967dea4135cb75490e1e801c4639efc"
+                    "versions.yml:md5,83d1b23f5ff5b2ad1b96d17d7d7594ee"
                 ],
                 "sra": [
                     [
@@ -28,7 +28,7 @@
                     ]
                 ],
                 "versions": [
-                    "versions.yml:md5,c967dea4135cb75490e1e801c4639efc"
+                    "versions.yml:md5,83d1b23f5ff5b2ad1b96d17d7d7594ee"
                 ]
             }
         ],
@@ -53,7 +53,7 @@
                     ]
                 ],
                 "1": [
-                    "versions.yml:md5,c967dea4135cb75490e1e801c4639efc"
+                    "versions.yml:md5,83d1b23f5ff5b2ad1b96d17d7d7594ee"
                 ],
                 "sra": [
                     [
@@ -67,7 +67,7 @@
                     ]
                 ],
                 "versions": [
-                    "versions.yml:md5,c967dea4135cb75490e1e801c4639efc"
+                    "versions.yml:md5,83d1b23f5ff5b2ad1b96d17d7d7594ee"
                 ]
             }
         ],
@@ -77,4 +77,4 @@
         },
         "timestamp": "2024-02-28T11:48:37.428307"
     }
-}
\ No newline at end of file
+}
diff --git a/subworkflows/nf-core/fastq_download_prefetch_fasterqdump_sratools/tests/main.nf.test.snap b/subworkflows/nf-core/fastq_download_prefetch_fasterqdump_sratools/tests/main.nf.test.snap
index 9972556a8f8..1a34e0b8af1 100644
--- a/subworkflows/nf-core/fastq_download_prefetch_fasterqdump_sratools/tests/main.nf.test.snap
+++ b/subworkflows/nf-core/fastq_download_prefetch_fasterqdump_sratools/tests/main.nf.test.snap
@@ -40,7 +40,7 @@
         "content": [
             [
                 "versions.yml:md5,1a2218ff913fc33408bffccb081b5048",
-                "versions.yml:md5,98d78bba9f3da39a0b7db6e9c7dcc224",
+                "versions.yml:md5,53d6e983afde3a28add2ffc6b7eba4f3",
                 "versions.yml:md5,9c558ff624585a6eee82a19c8c0136db"
             ]
         ],
@@ -94,4 +94,4 @@
         },
         "timestamp": "2024-02-28T12:02:56.161354"
     }
-}
\ No newline at end of file
+}