Background: Deoxycytidine kinase (DCK) is responsible for activation of gemcitabine by phosphorylation of gemcitabine to its monophosphate form. Studies have suggested that DCK expression may be correlated to response to gemcitabine in various solid tumors, and that downregulation of DCK may be at least partly responsible for gemcitabine-resistance in vitro. However, the significance of DCK expression in sarcomas as a biomarker of response to gemcitabine remains poorly characterized. Methods: Participants enrolled in the prospective Electronic Health Record (EHR)-Based Comprehensive Bone and Soft Tumor Registry (NCT02677961) at The Ohio State James Cancer Center who had received gemcitabine-based chemotherapy for the treatment of bone and soft tissue sarcoma were eligible for this study. A total of 179 patients were identified who had received at least one cycle of gemcitabine-based chemotherapy, between January 1st, 2014, to December 31^st, 2022. Whole exome RNASeq expression data was available for 36 of these patients who had received gemcitabine-based chemotherapy in the setting of active disease. Results: Of the 36 patients included in this study, 22 were female and 14 were male. Nine different main disease types were identified in the patient subset. These include angiosarcoma (8), chondrosarcoma (1), leiomyosarcoma (16), liposarcoma (3), malignant peripheral nerve sheath tumor (1), myxofibrosarcoma (2), osteosarcoma (2), undifferentiated pleomorphic sarcoma (3), and undifferentiated spindle cell sarcoma (1). The median time to treatment discontinuation (TTD) among all patients was 178 days. Among DCK low-expressors (n = 16), the median TTD was 86 days; among high-expressors (n = 16), the median TTD was not reached. Differences between groups did not reach statistical significance (HR = 0.55, p = 0.14). Conclusions: DCK expression by clinical RNASeq is a potential biomarker of gemcitabine-response in sarcoma, but larger studies are necessary to evaluate the potential clinical significance and possible role of DCK expression in identifying patients most likely to benefit from gemcitabine-based chemotherapy.