The aim of this study was to detect new polymorphisms in the bovine β-casein (β-CN) gene and to evaluate association of (new) β-CN protein variants with milk production traits and milk protein composition. Screening of the β-CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of β-CN protein variant I in the Dutch HF population. Studies of association of β-CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of β-CN protein variants with milk composition was studied in 1857 first-lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. β-CN protein variant I was significantly associated with protein percentage and protein yield, and with αs1-casein (αs1-CN), αs2-casein (αs2-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), casein index and casein yield. Inferring β-κ-CN haplotypes showed that β-CN protein variant I occurred only with κ-CN variant B. Consequently, associations of β-κ-CN haplotype IB with protein percentage, κ-CN, α-LA, β-LG and casein index are likely resulting from associations of κ-CN protein variant B, while associations of β-κ-CN haplotype IB with αs1-CN and αs2-CN seem to be resulting from associations of β-CN variant I.