The objective of this study was to determine the effect of cow genetic strain on fatty acid (FA) profiles in adipose tissue and milk. Adipose samples from two subcutaneous (shoulder and tail-head) and three visceral (kidney channel, mesenteric and omental) depots were obtained post mortem from New Zealand (NZ; n = 8) and North American (NA; n = 8) Holstein-Friesian cows. At the time of slaughter cows were in similar body condition (NZ: 4.0 ± 0.03, NA: 4.0 ± 0.02; ± SD) and stage of lactation (NZ: 90 ± 11.2 d; NA: 83 ± 4.3 d; ± SD). Milk was collected during the a.m. milking prior to slaughter and milk fat was extracted. Adipose and milk fat FA were quantified using gas chromatography. NZ cows had a lower proportion of saturated FA in shoulder, tail-head and omental adipose tissue and a greater proportion of mono-unsaturated FA and an elevated Δ9-desaturase index in shoulder and tail-head adipose tissue. The proportions of individual FA differed between adipose depots, with proportions of de-novo FA greater in subcutaneous compared with visceral adipose depots. Milk from NZ cows contained greater concentrations of short chain FA (C8 : 0-12 : 0) and CLA, and less cis-9 18 : 1 than milk from NA cows. Regression analysis identified moderate associations between milk FA and shoulder adipose tissue FA for 18 : 2 (R(2) = 0.24), 18 : 3 n - 3 (R(2) = 0.39), and polyunsaturated fatty acids (R(2) = 0.38). Results from this study support the hypothesis that genetic strain dictates FA profiles in adipose tissue and milk and may alter the metabolic status of the various adipose depots differently. The data further support the premise that genetic strain affects the metabolic status of the various adipose depots differently. Elucidating the mechanisms that regulate the different adipose depots in the NZ and NA strains will increase our understanding of tissue mobilization and replenishment.