Some dietary fatty acids (FA) may influence adipose tissue FA composition. The objective of this study was to compare the long-term effects of late lactation fat feeding on subcutaneous adipose and plasma FA profiles during the peripartum period in multiparous (MP) cows. To determine if there is an influence of mobilization cycle, a secondary objective was to compare MP and primiparous (PP) adipose tissue FA profile. Multiparous cows were assigned to either a C16 (pure palmitic acid + concentrate pellet) or C18 (concentrate pellet with 50% FA [78% C18, 22% C16]) supplemented diet (2.3% DM added FA) for the last 50 ± 8 d of late lactation. Cows were pen fed (2/treatment) until dry off and then combined during the dry period. At −31 (±1) days relative to calving (DRTC), MP cows were cohoused with PP cows. Subcutaneous adipose biopsies were taken from MP cows before enrollment, and from all cows at −31 DRTC. Blood samples were taken at −31 and 5 DRTC. Fatty acid profiles were determined via GC. Data were analyzed in SAS 9.4 using PROC MIXED with a fixed effect of treatment (combination of parity and dietary fat; experimental units n = 6), with preplanned contrasts of: MP vs. PP, and MP C16 vs. MP C18. There was no effect of diet on MP cow adipose FA profile at enrollment or −31 DRTC (P > 0.10), but MP cows had greater C16:0 (27.4 vs. 25.3 ± 0.4 g/100g FA; P = 0.04) and less C18:0 (10.3 vs. 12.5 ± 0.6 g/100g FA; P = 0.08) and C18:2 (1.6 vs. 2.6 ± 0.1 g/100g FA; P < 0.01) than PP cows at −31 d. At 5 DRTC, MP cows had increased plasma C16:0 (18.2 vs. 16.2 ± 0.3 g/100g FA; P = 0.04) and C18:1 (20.7 vs. 15.4 ± 0.7 g/100g FA; P = 0.04) but decreased C18:0 (16.2 vs. 18.6 ± 0.3 g/100g FA; P = 0.03) in agreement with increased (P < 0.05) NEFA. The difference in dietary FA profile during late lactation was either not great enough to alter subcutaneous adipose tissue profile, or adipose tissue metabolism regulated the profile stored. The difference between MP and PP animals suggests that over lactations, there are influences of diet and mobilization cycles on adipose tissue FA profile in MP animals.