Background Significant associations between total nonesterified fatty acid (NEFA) concentrations and incident stroke have been reported in some prospective cohort studies. We evaluated the associations between incident stroke and serum concentrations of nonesterified saturated, monounsaturated, polyunsaturated, and trans fatty acids. Methods and Results CHS (Cardiovascular Health Study) participants (N=2028) who were free of stroke at baseline (1996–1997) and had an archived fasting serum sample were included in this study. A total of 35 NEFAs were quantified using gas chromatography. Cox proportional hazards regression models were used to evaluate associations of 5 subclasses (nonesterified saturated, monounsaturated, omega (n)‐6 polyunsaturated, n‐3 polyunsaturated, and trans fatty acids) of NEFAs and individual NEFAs with incident stroke. Sensitivity analysis was conducted by excluding cases with hemorrhagic stroke (n=45). A total of 338 cases of incident stroke occurred during the median 10.5‐year follow‐up period. Total n‐3 (hazard ratio [HR], 0.77 [95% CI, 0.61–0.97]) and n‐6 (HR, 1.32 [95% CI, 1.01–1.73]) subclasses of NEFA were negatively and positively associated with incident stroke, respectively. Among individual NEFAs, dihomo‐γ‐linolenic acid (20:3n‐6) was associated with higher risk (HR, 1.29 [95% CI, 1.02–1.63]), whereas cis‐7‐hexadecenoic acid (16:1n‐9c) and arachidonic acid (20:4n‐6) were associated with a lower risk (HR, 0.67 [95% CI, 0.47–0.97]; HR, 0.81 [95% CI. 0.65–1.00], respectively) of incident stroke per standard deviation increment. After the exclusion of cases with hemorrhagic stroke, these associations did not remain significant. Conclusions A total of 2 NEFA subclasses and 3 individual NEFAs were associated with incident stroke. Of these, the NEFA n‐3 subclass and dihomo‐γ‐linolenic acid are diet derived and may be potential biomarkers for total stroke risk.