The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the alpha3 and transmembrane domains is unprecedented.