Background: Despite novel therapies for relapsed/refractory (R/R) DLBCL, outcomes among this cohort remain poor. A prior study indicated that patients who lost CD20 expression after treatment have earlier disease progression, but whether this translates to inferior outcomes remains unknown. We hypothesized that R/R DLBCL patients who lost CD20 expression upon disease relapse would have worse survival and response rates compared to those who retained expression. Methods: We performed a retrospective cohort study of R/R DLBCL patients seen at the University of Michigan between 2007-2021. Inclusion criteria were patients ≥ 18 years old, diagnosis of CD20 positive (+) DLBCL (de novo or transformed), relapsed/refractory disease, and CD20 status available at relapse on flow cytometry/immunohistochemistry. Overall survival (OS) was defined as time from first relapse to death and relapse-free survival (RFS) was time from first relapse to death or next treatment; both of which were estimated with Kaplan Meier analysis. Overall response rate (ORR) was complete response or partial response after first salvage therapy. Descriptive statistics with univariate and multivariate analyses were also performed. Results: 98 patients were included (65% male) with median age at diagnosis of 62 years. Majority of patients had stage III/IV disease at diagnosis (85%), 45% with GCB subtype and 7% with high-grade lymphoma. At diagnosis, all patients had CD20+ disease. Median time from diagnosis to first relapse was 7 months and median time from completion of induction treatment to first relapse was 2.4 months. Among the 98 patients, 84 were CD20+ at first relapse, 12 were CD20 negative and the remaining 2 patients were indeterminate and excluded from survival analyses. The estimated OS in the CD20+ group was 17.9 months (95% CI 14.0, 41.8) compared to 10.6 months in the CD20 negative group (95% CI 6.9, Not Reached). Although median OS in the CD20+ group was longer, a two-sample log-rank test failed to identify a significant difference in OS between the two groups (p = 0.085). The estimated RFS in the CD20+ group was 3.5 months (95% CI 3.3, 3.9) compared to 4.4 months in the CD20 negative group (95% CI 2.6, Not Reached, p = 0.739). ORR among CD20+ and CD20 negative patients was 64% and 50%, respectively. Of patients who became CD20 negative at relapse, 100% received anti-CD20 directed therapy thereafter, as part of salvage or with conditioning prior to transplant. Conclusions: Patients with DLBCL who relapse and maintain CD20 positivity seem to have a trend towards longer OS, though this was not statistically significant in our analysis. Our study was limited given the retrospective data collection and small sample size which may have limited our ability to detect a statistical significance in outcomes. In the future, we will plan to expand our study to involve other academic institutions and reassess outcomes among these cohorts.