Taxonomic uncertainties and morphologic and molecular similarities may lead to incorrect or incomplete species identifications of immature ticks. This problem can be compounded when the diagnostic morphological features used are unreliable, unstable, or difficult to view. Our study attempts to sort out the similar morphologies of three Amblyomma spp. nymphs with partially overlapping geographic distributions in South Texas. Amblyomma americanum (L.), Amblyomma mixtum Koch, and Amblyomma tenellum Koch are three closely related species with similar nymphs and larvae. Previous attempts to morphologically distinguish among nymphs of these species have relied on difficult-to-observe and unreliable characters. Over the course of decades of routine diagnostic work with these ticks, we have observed and discovered consistent and useful morphologic features sufficient to practically distinguish among these nymphs. We confirmed our morphological studies with molecular genetic evidence (i.e., 12S mitochondrial genes), and we propose that practical, rapid morphological species-level identification of nymphs of the three named species is now possible with confidence.