Fluorescence in situ hybridization (FISH) was used to identify which chromosome pairs are involved in the difference in karyotypes of three clonal lines of Rainbow Trout (Oncorhynchus mykiss): OSU with 2n = 60, Clearwater with 2n = 58, and Swanson with 2n = 58. These lines appear to have karyotypes typical of strains from the regions from which they were derived (California, Idaho, and Alaska, respectively). Bacterial artificial chromosome clones containing mapped markers specific for different acrocentric pairs in the OSU strain were hybridized to chromosomes of hybrid fish. The OSU × Clearwater and OSU × Swanson hybrids have 2n = 59 with a chromosome pair consisting of one metacentric (fusion) chromosome and two acrocentrics. Both strains have the same chromosome fusion involving acrocentric chromosomes 25 and 29 of the OSU strain although they are from widely separate geographic regions. These results suggest that all of the 2n = 58 Rainbow Trout may have the same karyotype and a common origin.