We investigated the levels and distribution of genetic diversity in two presumed sister species of milkweeds: the widespread Asclepias perennis and the narrowly distributed A. texana. Horizontal starch-gel electrophoresis was performed on leaf tissue from 942 individuals from 18 populations of A. perennis and 357 individuals representing 11 populations of A. texana from throughout their respective ranges. All 16 loci were polymorphic in A. perennis, with a mean of 3.63 alleles per locus at the species level, and 1.57 +/- 0.29 (mean +/- standard error) alleles per locus at the population level. In A. texana 15 loci were polymorphic, with a species average of 3.00 alleles per locus and 1.51 +/- 0.17 alleles per locus at the population level. Overall species-level mean heterozygosity was slightly higher in A. perennis (H(es)BAR = 0.076 +/- 0.035) than A. texana (H(es)BAR = 0.064 +/- 0.020). Mean expected heterozygosity within populations was the same for both species (H(es)BAR = 0.061 +/-0.009). Total gene diversity was low (A. perennis, H(T)BAR = 0.076; A. texana, H(T)BAR = 0.069), with most of this variation found within populations (A. perennis, H(S)BAR = 0.058; A. texana, H(S)BAR = 0.060). Among-population diversity relative to total diversity was very low (A. perennis, G(ST)BAR = 0.082; A. texana, G(ST)BAR = 0.068). Genetic identities within species averaged 0.95 for A. perennis and 0.98 for A. texana, but these taxa were quite distinct from each other (IBAR = 0.79). These species were even more distinct from another milkweed, A. exaltata (for A. perennis, IBAR = 0.63; for A. texana, IBAR = 0.39). Using several lines of evidence, we estimate that A. perennis and A. texana diverged 1-2 x 10(6) years B.P.