The current study investigated associations between variation in the bovine perilipin-2 gene (PLIN2) and milk traits (milk fat content, milk protein content, milk yield and milk fatty acid (FA) component levels) in 409 New Zealand pasture-grazed Holstein-Friesian × Jersey-cross (HF × J-cross or Kiwicross™) cows. Five nucleotide sequence variants were found in three regions of the gene, including c.17C>T in exon 2, c.53A>G in exon 3, c.595+23G>A and c.595+104_595+108del in intron 5, and c.*302T>C in the 3′-untranslated region. The c.*302T>C substitution produces two nucleotide sequence variants (A5 and B5), and this variation was associated with variation in milk protein content and milkfat composition for C10:0, C11:0, C12:0, C13:0 and C16:0 FA and medium-chain fatty acid (MCFA) and long-chain fatty acid (LCFA) groups. After correcting for the effect of variation in the diacylglycerol acyl-CoA acyltransferase 1 gene (DGAT1) that results in the amino acid substitution p.K232A, variation in the FA binding protein 4 gene (FABP4) and variation in the stearoyl-CoA desaturase (Δ-9-desaturase) gene (SCD) that results in the amino acids substitution p.A293V, significant differences between A5A5 and B5B5 cows were found for C10:0, C11:0, C12:0, C13:0, C16:0, and the MCFA, LCFA, total saturated FA and C10:1 index groups. This suggests that nucleotide sequence variation in PLIN2 may be affecting milk FA component levels.