Knowledge on genetic polymorphisms of metabolising enzymes including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP3A131, one of the major CYP isoforms in the feline liver and small intestine. Eight non-synonymous variants and one synonymous variant of feline CYP3A131 were identified in 29 cats. A major non-synonymous type was not observed. Metabolic parameters (Km and Vmax) of dibenzylfluorescein hydroxylation were ranged within about 2 times for the identified non-synonymous variants by using a heterologous coexpression system of CYP3A131 and feline cytochrome P450 reductase in Escherichia coli. The results confirmed the polymorphic nature of CYP3A131 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats.