Chagas disease is one of the most important vector-borne diseases in Mexico. Triatoma pallidipennis (Stål) is one of the most epidemiologically important vector species. Despite being classified as a single species, various studies (molecular, morphometric, and biological) on populations across its distribution suggested it is composed of a group of cryptic species. This study examined reproductive isolation among 5 populations of T. pallidipennis originating from the western, southern, and central regions of Mexico to help clarify the potential existence of a cryptic species complex of T. pallidipennis in Mexico. A generation of hybrids was analyzed for fertility and fecundity. Fertility rates varied from 50% to 100% in the parental crosses and from 20% to 100% in the F1 × F1 crosses. Fecundity ranged from 1.4 to 3.2 eggs/female/day in the parental crosses, which decreased to 0.9–2.9 in the F1 × F1 crosses (except in Jalisco × Morelos). The fertility of the eggs ranged from 61.4% to 85.4% in the parental crosses, dropping to 44% to 90.1% in some F1 × F1 crosses, indicating partial reproductive isolation among these populations.