The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P, G1P, G2P, G3P, G3P, G3P, G4P, G4P, G6P, G6P, G6P, G8P, G8P, G9P, G9P, G10P, G12P and G12P) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P), NCDV (bovine, G6P), K9 and CU-1 (canine, G3P), together with 31 field isolates (canine, G3P; feline, G3P; porcine, G2P, G3P, G4P, G5P, G5P, G5P, G5P, G9P and G9P) were also successfully NSP4-typed. Four human G3P strains and one feline G3P strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.