Trapdoor spiders (Bothriocyrtum californicum) from nine southern California sites, including three on Santa Catalina and Santa Cruz Islands, were compared, based on variation at nine allozyme loci. Populations were generally in Hardy-Weinberg equilibrium, with no evidence of inbreeding. Levels of genetic variability in the six mainland populations were relatively high (mean H_o = 10.8%, mean P = 42.6%), while it was reduced (H_o < 2.0%, P < 23.0%) in two of three island populations, consistent with the loss of variability usually associated with island colonization. Genotype frequency distributions indicated a recent reduction in effective population size for spiders from Webb School of California, perhaps a consequence of habitat destruction associated with school construction in 1920–1930. Interpopulation differentiation was generally substantial and did not bear a simple relationship to physical distance. These findings suggest that, while B. californicum populations generally possess ample levels of genetic variability relative to other invertebrates, bottlenecks do occur, and populations are usually genetically isolated. To foster preservation of the existing gene pool, management of B. californicum should focus on maintaining as many populations in situ as possible and facilitating connections between them, while also creating or restoring habitat for potential colonization.