Dietary fat, including specific fatty acids (FAs), has been proposed to contribute to prostate cancer pathogenesis, but findings from studies based on biomarkers have been conflicting. We examined the association between erythrocyte membrane FA composition and prostate cancer risk in a nested case‐control study within a large cohort of African Americans, Native Hawaiians, Japanese Americans, Latinos, and Whites. Analyses included 376 cases and 729 matched controls. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) with adjustment for the matching criteria of ethnicity, age and date of blood draw, and fasting status, as well as family history of prostate cancer, BMI, physical activity, and education. No significant association was found for saturated, mono‐ and polyunsaturated FAs, or for specific n‐3 and n‐6 FAs, even when the analysis was limited to advanced or high grade prostate cancer. An ethnic‐specific analysis of African Americans, the only group with enough case numbers, showed a suggestive, but not statistically significant inverse association between α‐linolenic acid and prostate cancer risk (OR for the highest vs. lowest tertile = 0.39, 95% CI = 0.14–1.09, Ptrend = 0.09). Our findings do not support an association between FAs and prostate cancer risk. Grant sponsor: National Cancer Institute; Grant No. R37 CA54281.