A novel PCR method was developed to discriminate amongst genotypes A-C of the rotavirus non-structural protein 4 (NSP4). Genotype-specific primers were designed that correctly identified the NSP4 genotype when evaluated as a multiplex PCR with cell culture adapted rotavirus strains. Rotavirus strains B223 SGIG6P6[1], NCDV SGIG6P6[1] and SA11 SGIG3P5B[2] were used as control for NSP4 genotype A; A34 SGIG5P14[23], Gottfried SGIIG4P2B[6] and Wa SGIIG1P1A[8] for NSP4 genotype B; RRV SGIG3P5B[3] for NSP4 genotype C. Subsequently, the same set of specific primers was used to genotype a set of 77 Swedish clinical samples. The results showed that all human clinical samples analyzed belong to the NSP4 genotype B and the VP6 subgroup II.