Strains of Chlamydomonas allensworthii Starr, Marner, and Jaenike isolated from 13 sites worldwide in distribution were compared to assess the congruity of DNA similarity, sexual behavior, and pheromone response type. The isolates fell into two pheromone response categories. One of these included isolates of widespread distribution in the United States, and all appeared to be capable of forming zygotes inter se. The second group was larger and more complex, encompassing four mating groups as defined by zygote formation. Comparison of DNA sequences (internal transcribed spacers 1 and 2 of the nuclear rDNA repeats) fully supported the same subdivision of the isolates. Both selfing and outbreeding clones were among the isolates. The possibility that other Volvocales species might respond to these same pheromones remains to be tested.