A persistent challenge is the lack of reliable serum diagnostic tests for peripheral arterial disease (PAD). Serum cholesterol levels lack sensitivity for PAD and are not predictive of disease progression. Our team previously demonstrated that serum circulating Fatty Acid Synthase (cFAS) correlates with incidence of PAD and does not correlate with serum lipids. Therefore, we hypothesized that cFAS can be an independent diagnostic biomarker for PAD in a larger cohort, with or without diabetes. An IRB-approved consecutive (2014-2018) cohort was used to identify individuals with PAD/diabetes, as well as healthy controls. Consented patients provided a serum sample, and total cFAS content was measured with ELISA. Demographics and incidence of PAD/diabetes were collected from chart review. Serum cFAS and demographics were compared, and regression was used to determine correlations with cFAS. A total of 165 patients met inclusion criteria. Of these, 61 (37%) had PAD and diabetes (Group 1), 64 (38.8%) had PAD and no diabetes (Group 2), and 40 (24.2%) were healthy controls (Group 3). Compared to Group 3, Groups 1&2 were significantly older, had more males, and higher incidence of cardiovascular co-morbidities (p<0.001). Compared to Group 2, Group 1 had a higher incidence of cardiovascular/cerebrovascular disease, CHF, and HTN (p<0.05). Despite these differences, both Groups 1&2 had significantly higher serum cFAS content than Group 3 (p<0.01 and p=0.01, respectively). In the whole cohort, there were significant correlations between cFAS and PAD severity and between cFAS and smoking status. (p=0.02, p=0.01 respectively). Our study suggests that cFAS can serve as an independent serum-based diagnostic biomarker for PAD, regardless of diabetes status. Future studies will further validate this serum biomarker and determine its predictive value for identifying individuals who are at highest risk of disease progression.