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The minute pirate bugs, Orius insidiosus (Say) and Orius pumilio (Champion) (Hemiptera: Heteroptera: Anthocoridae), are closely related species known to be sympatric in north Florida. Here, male and female genitalia, DNA sequences, and the effects of within- and between-species pairings on egg production and egg development were examined to develop a better understanding of the relationship between these two species. Interspecific matings between the two species did not result in viable progeny. Although there were gross similarities in the morphology of the male parameres (external genitalia) between the two species, the cone in O. pumilio was much broader with a greater spiral twist and the flagellum was longer than in O. insidiosus. Correspondingly, there were differences in the morphology of the copulatory tubes of the females of the two species. In O. insidiosus, the organ was somewhat longer than in O. pumilio and oriented parallel to the abdominal midline, while the copulatory tube in O. pumilio tilted slightly towards the midline. Additionally, the copulatory tube for O. pumilio included a sclerotized basal mound that was not present in O. insidiosus. These morphological differences suggest that successful copulation between these species could be difficult. In contrast to conspecific matings, interspecific matings resulted in few or no eggs laid over a period of two weeks and no viable progeny. Comparison of the 18S ribosomal gene ITS-1 sequences between the two species demonstrated only 91% homology. When yolk protein contents were examined to determine whether reproductive physiology had shifted to full egg production, interspecifically mated females contained amounts of yolk protein comparable to that in fed, but unmated females; this was less than 10% of the yolk protein previously found in fed and conspecifically mated females. These findings together confirm that O. insidiosus and O. pumilio are indeed two separate species.