Plasma and liver fatty acid (FA) profiles in transition cows are influenced by the degree of FA mobilization from adipose tissue which is often exacerbated in cases of hyperketonemia (HYK). The objectives were to examine the liver and blood FA profiles of cows diagnosed with HYK (≥1.2 mM BHB) or not (nonHYK). Liver biopsies were taken at +1, +14, and +28 and blood samples at −28, +1, +3, +7, +11, +14, and +28 d relative to calving (DRTC). Liver and plasma total FA were acid methylated and analyzed using gas chromatography. Data were analyzed using PROC MIXED and PROC CORR (SAS 9.4). The model contained the fixed effects of block, HYK diagnosis, DRTC, the interaction of HYK diagnosis and DRTC, and the random effect of cow using repeated measures. PROC CORR was used to determine correlations between liver and plasma FA within DRTC and FA. Comparisons between HYK and nonHYK are expressed as mean ± SE. Plasma of cows diagnosed with HYK had an increased proportion of C18:1 (18.2 vs. 16.7 ± 0.5 g/100g FA; P = 0.02) and decreased proportion of C18:2 (36.2 vs. 38.7 ± 0.6 g/100g FA; P = 0.01). Plasma C16:0 and C18:0 were not different by HYK. Reflective of plasma differences by HYK status, liver of cows diagnosed with HYK had an increased proportion of C18:1 (27.0 vs. 25.3 ± 0.9 g/100g FA; P < 0.04) and decreased proportion of C18:2 (10.2 vs. 11.4 ± 0.5 g/100g FA; P < 0.10). Conversely, liver C16:0 was increased (29.6 vs. 27.0 ± 1.4 g/100g FA; P < 0.09) and C18:0 was decreased (15.5 vs. 17.9 ± 1.4 g/100g FA; P < 0.08) in cows with HYK. Correlations between liver and plasma FA proportions varied dependent on HYK diagnosis for C16:0 (r = 0.35, 0.44, 0.30 vs. 0.67, 0.21, 0.33 for HYK and nonHYK), C18:0 (r = 0.20, 0.41, −0.19 vs. 0.86, −0.08, −0.33) and C18:1 (r = 0.74, 0.84, 0.58 vs. 0.62 −0.25, 0.63) at 1, 14 and 28 DRTC. Generally, C18:2 was similar between HYK and nonHYK at all time points (r = 0.35, 0.44, 0.30 vs. 0.67, 0.21, 0.33). These data suggest that FA profiles during mobilization may be shifted in cows with HYK and may influence downstream FA profiles of deposited liver lipids. Potential influences on preferential liver FA oxidation or storage during metabolic challenge should be further examined.