Understanding of the aetiology of congenitally anomalous pulmonary valves remains incomplete. The aim of our study, therefore, was to elucidate the degree to which the phenotypic variation known to exist for the pulmonary valve relies on genotypic variation. Initially, we tested the hypothesis that genetically alike individuals would display similar valvar phenotypes if the phenotypic arrangement depended entirely, or almost entirely, on the genotype. Thus, we examined pulmonary valves from 982 Syrian hamsters belonging to two families subject to systematic inbreeding by crossing siblings. Their coefficient of inbreeding was 0.999 or higher, so they could be considered genetically alike. External environmental factors were standardized as much as possible. A further 97 Syrian hamsters from an outbred colony were used for comparative purposes. In both the inbred and outbred hamsters, we found valves with a purely trifoliate, or tricuspid, design, trifoliate valves with a more or less extensive fusion of the right and left leaflets, bifoliate, or bicuspid, valves with fused right and left leaflets, with or without a raphe located in the conjoined arterial sinus, and quadrifoliate, or quadricuspid, valves. The incidence of the different valvar morphological variants was similar in the outbred and inbred colonies, except for the bifoliate pulmonary valves, which were significantly more frequent in the hamsters from one of the two inbred families. Results of crosses between genetically alike hamsters revealed no significant association between the pulmonary valvar phenotypes as seen in the parents and their offspring. The incidence of bifoliate pulmonary valves, nonetheless, was higher than statistically expected in the offspring of crosses where at least one of the parents possessed a pulmonary valve with two leaflets. Our observations are consistent with the notion that the basic design of the pulmonary valve, in terms of whether it possesses three or two leaflets, relies on genotypic determinants. They also denote that the bifoliate condition of the valve is the consequence of complex inheritance, with reduced penetrance and variable expressivity. Moreover, in showing that the incidence of the bifoliate pulmonary valve significantly differs in two different isogenetic backgrounds, our data suggest that genetic modifiers might be implicated in directing the manifestation of such specific pulmonary valvar malformations. Finally, our findings indicate that factors other than the genotype, operating during embryonic life and creating developmental noise, or random variation, play a crucial role in the overall phenotypic variation involving the pulmonary valve.