Japanese pear (Pyrus pyrifolia) exhibits gametophytic self-incompatibility (GSI) controlled by a complex and multiallelic S locus. The pistil-part product of the S locus is the polymorphic ribonuclease, S-RNase. Information on S-genotypes is important for the production and breeding of Japanese pears. Molecular analyses of S-genotypes of Japanese pear have been conducted with the CAPS (cleaved amplified polymorphic sequence) system; PCR amplification of S-RNase fragments by a common primer pair followed by digestion with restriction enzymes each of which cleaves a specific S haplotype. Here, we show that the separation of S-RNase fragments by polyacrylamide gel electrophoresis (PAGE) distinguishes four out of nine S haplotypes of Japanese pear without restriction digestion. S(3)-, S(5)-, S(6)- and S(8)-RNases were identified as distinct bands by PAGE. S(3)- and S(5)-RNases were separated by PAGE despite their identical fragment sizes. Using this system, three Japanese pear lines with unknown S-genotypes were analyzed. The newly determined S-genotypes of the lines were confirmed by CAPS analysis.