BACKGROUND: Synergistic or additive effects or both on cardiometabolic risk may be missed by examining individual fatty acids (FAs). A pattern analysis may be a more useful approach. In addition, it remains unclear whether erythrocyte FA composition relates to insulin resistance among Hispanics/Latinos. OBJECTIVE: We derived erythrocyte FA patterns for a Puerto Rican cohort and examined their association with diet and insulin resistance in cross-sectional and prospective analyses. DESIGN: At baseline, principal components analysis was used to derive factor patterns with the use of 24 erythrocyte FAs from 1157 participants of the Boston Puerto Rican Health Study (aged 45-75 y). Dietary intake was assessed with a validated semiquantitative food-frequency questionnaire. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated at baseline and at the 2-y follow-up. Relations between FA patterns and HOMA-IR were analyzed in a sample of 922 participants with available data. RESULTS: Five FA patterns were derived, differentiated by 1) relatively high de novo lipogenesis (DNL) FAs and low n-6 (ω-6) FAs, 2) high very-long-chain saturated FAs, 3) high n-3 (ω-3) FAs, 4) high linoleic acid and low arachidonic acid, and 5) high trans FAs. The DNL pattern was positively correlated with sugar and inversely with n-6 and monounsaturated FA intakes. Only the DNL pattern was positively related to baseline HOMA-IR [adjusted geometric means (95% CIs) for quartiles 1 and 4: 1.72 (1.58, 1.87) and 2.20 (2.02, 2.39); P-trend < 0.0001]. Similar associations were observed at 2 y, after adjustment for baseline status [quartiles 1 and 4 means (95% CIs): 1.61 (1.48, 1.76) and 1.84 (1.69, 2.00); P-trend = 0.02]. These results remained consistent after the exclusion of participants with diabetes (n = 485). CONCLUSION: Our findings suggest that upregulated DNL associated with a diet high in sugar and relatively low in unsaturated FAs may adversely affect insulin sensitivity in a Hispanic/Latino cohort.