Analysis of C-band heterochromatin (CBH) and cold-induced secondary constrictions (CICs) indicates that gynogenetic triploidy in the Ambystoma jeffersonianum complex is a cytogenetic consequence of hybridization between the two diploid species, A. jeffersonianum and A. laterale. The key feature in the history of this complex was the apparent proclivity for germ-line chromosome reduplication, and incidental production of chromosomally unreduced ova, allowing the circumvention of meiotic difficulties in diploid hybrid females. Chromosome structure, in terms of CBH and CICs, the mechanism of sex determination (dominant W, female heterogametic), and a recognizable WZ female/ZZ male sex chromosome heteromorphism in the diploid species A. laterale, are cytogenetic factors that allow reconstruction of the probable evolutionary history of the complex. The constitution of the triploid karyotypes suggests that the putative ancestral hybrid diploid females resulted from a mating between female A. jeffersonianum and male A. laterale, and that when such a hybrid female backcrossed to normal males of A. jeffersonianum and A. laterale, both kinds of allotriploids, A. platineum and A. tremblayi respectively, were produced. Karyological differentiation in each triploid species suggests that their origin was relatively recent and virtually simultaneous. It is conceivable that only one such hybrid female gave rise to both kinds of allotriploids in just one or two breeding seasons, and that present geographic distributions are due to persistent post-glaciation migrations of the resulting triploid clones. All offspring from such a back-cross carried a jeffersonianum W-chromosome and hence were female as well as triploid, and probably continued to produce chromosomally unreduced (triploid) ova by the same mechanism that operated in their hybrid mother. Sperm rejection resulting in gynogenesis in the allotriploids can be viewed as a physiological response to “pseudopolyspermy” by the chromosomally unreduced triploid ova. Evidence is presented that one of the triploid species, A. platineum, may produce a high percentage of diploid offspring with karyotypes identical to A. jeffersonianum. Diploids have not been detected among the offspring of A. tremblayi, but tetraploids are occasionally produced.