This short communication aimed to develop a fast and straightforward method for the simultaneous discrimination in buffaloes of the alleles CSN1S1 A/B and CSN3 X1/X2 using a single protocol. DNA was isolated from 219 individual blood samples. A duplex artificially created restriction site (ACRS-PCR) was accomplished using two pairs of primers generating 86 bp (CSN1S1) and 160 bp (CSN3). Amplicons were contemporary digested by MboI and HinfI for the identification of genotypes. The double simultaneous amplification and digestion proved to be effective for allele identification. The method resulted particularly quick due to the small PCR amplicons and fast digest enzymes that allowed both a rapid amplification (about 1 h and 40 min) and digestion in 10 min. Population analysis indicated that minor allele frequencies were CSN1S1 A (0.425) and CSN3 X2 (0.306). Linkage disequilibrium (LD) showed r 2 ¼0.46, and haplotype analysis revealed all four possible combinations with higher frequency for the CSN1S1 B-CSN3 X1 (0.553). Considering the tremendous economic impact of the CSN1S1-CSN3 variants on the dairy production in buffalo, this method, applicable immediately after the birth from any DNA source, may speed up the selection of sires and dams' lines with more favourable genotypes. HIGHLIGHTSCSN1S1-CSN3 composite genotype influences dairy performances. Duplex ACRS-PCR identifies the CSN1S1-CSN3 composite genotype in a single protocol. The duplex ACRS-PCR method may speed up the selection of lines with more favourable genotypes.