Laboratory and experimental studies of four of the five subspecies of Euxoa comosa (Morr.), i.e. annir, altera, lutulenta and Ontario, each formerly considered of specific rank, were undertaken to assess their taxonomic status. Morphologically the immature stages of the subspecies are similar although lutulenta has a 40–50% larger egg and the larvae of lutulenta and Ontario are darker than those of annir and altera. The subspecies show differentiation in larval growth rate, number of larval instars, duration of preimaginal development and length of the preoviposition period. The possible adaptive significance of these differences are discussed. Hybrid F1 were obtained from 11 of the 12 possible pairwise combinations. Eight of the F1 hybrids were inbred and six produced fertile eggs. Some of the F2 progenies exhibited a wide range of phenotypes encompassing all subspecies including those of the 5th subspecies, E. c. comosa. Although hybridization success was high there was some evidence of genetic incompatibility including dyssynchronous emergence of the sexes and the absence, shortage, or inviability of female moths in some crosses. Mating discrimination tests show a moderate level of mating bias between subspecies. Calling periods of females of the subspecies largely overlap although differences in the time at which calling begins might be responsible for some of the mating bias. Both laboratory and field tests of sex pheromone attraction indicate considerable sex pheromone specificity among the subspecies. The results are discussed with reference to a companion paper which on the basis of conventional taxonomic characters and biogeographic considerations concludes that Euxoa comosa is best regarded as a polytypic species encompassing five subspecies.