Extensive reduction of the size and complexity of the pelvic skeleton (i.e., pelvic reduction) has evolved repeatedly in Gasterosteus aculeatus. Asymmetrical pelvic vestiges tend to be larger on the left side (i.e., left biased) in populations studied previously. Loss of Pitx1 expression is associated with pelvic reduction in G. aculeatus, and pelvic reduction maps to the Pitx1 locus. Pitx1 knockouts in mice have reduced hind limbs, but the left limb is larger. Thus left-biased directional asymmetry of stickleback pelvic vestiges may indicate the involvement of Pitx1 in pelvic reduction. We examined 6,356 specimens from 27 Cook Inlet populations of G. aculeatus with extensive pelvic reduction. Samples from 20 populations exhibit the left bias in asymmetrical pelvic vestiges expected if Pitx1 is involved, and three have a slight, non-significant left bias. However, samples from three populations have a significant right bias, and one large sample from another population has equal frequencies of specimens with larger vestiges on the left or right side. A sample of fossil threespine stickleback also has significantly left-biased pelvic vestiges. These results suggest that silencing of Pitx1 or the developmental pathway in which it functions in the pelvis is the usual cause of pelvic reduction in most Cook Inlet populations of G. aculeatu, and that it caused pelvic reduction at least 10 million years ago in a stickleback population. A different developmental genetic mechanism is implicated for three populations with right-biased pelvic vestiges and for the population without directional asymmetry. J. Exp. Zool. (Mol. Dev. Evol.) 306B, 2006. © 2006 Wiley-Liss, Inc.