The characterization of casein polymorphism is an essential step in order to understand the genetic basis of milk quality in dairy ruminants. In this work, we report the identification of a regulatory mutation at the buffalo αs1-casein (CSN1S1) gene that alters the normal processing of the primary transcript. Sequencing of CSN1S1 cDNA from individuals harbouring this new variant revealed that its most distinctive feature is the loss of exon 6 that encodes eight amino acids between positions 35-42 of mature protein. In an effort to map the causal mutation, we sequenced a genomic region spanning exons 5-7 of the buffalo CSN1S1 gene. This experiment allowed us to establish that exon 6-skipping is produced by a G to C substitution at the first position of intron 6 that inactivates the donor splice site. This mutation can be typed by PCR-RFLP by using either TaaI or Bpu10I diagnostic restriction enzymes, and it has a frequency of 0.18 in Romanian buffaloes. This exon skipping phenomenon is the first one described in buffalo CSN1S1 locus.