Background <p>CD5 positive diffuse large B-cell lymphoma (DLBCL) has unique clinical and pathological characteristics.</p> Methods <p>We analyzed 25 cases of CD5-positive DLBCL, focusing on their clinical, morphological, immunohistochemical and genetic features.</p> Results <p>Among the 13 females and 12 males, 14 were over 60 years (14/25, 56%). A majority of them presented in stage IV (15/21, 71.43%), primarily with extranodal lesions (14/25, 56%). The positive expression rates of CD20, CD10, BCL6, MUM1, MYC, and BCL2 were 100% (25/25), 40% (10/25), 84% (21/25), 100% (25/25), 87.5% (21/24) and 100% (25/25), respectively. Twenty-one cases (87.5%, 21/24) were MYC/BCL2 double-expressing. More than half of the cases were non-germinal center origin (15/25, 60%). The accuracy of p53 in predicting TP53 mutation was 95.65% (22/23). Genetic subtypes were MCD (13/25, 52%), TP53^Mut (7/25, 28%), EZB (1/25, 4%) and Other subtype (4/25, 16%). Among CD5 strong positive cases, 7 of the p53 wild-pattern cases were MCD subtype (7/8, 87.5%) and 6 of the p53 mutant-pattern cases were classified as TP53^Mut subtype (6/7, 85.71%). In the CD5 weak-moderate positive group, one p53 mutant-pattern case was TP53^Mut subtype (1/2, 50%), 4 of the wild-pattern cases were Other subtype (4/6, 66.67%).</p> Conclusions <p>For CD5 strong positive cases, a wild pattern of p53 likely indicates MCD subtype, while a mutant p53 pattern may suggest TP53^Mut subtype. CD5 and p53 may potentially offer initial molecular subtyping for CD5-positive DLBCLs. We suggest incorporating these two markers into the routine pathological diagnosis of DLBCL to assist in guiding preliminary classification and influencing treatment decisions.</p>