The mayaguana triad of the Drosophila repleta species group includes D. mayaguana, D. straubae, and D. parisiena, the latter two of which are very similar when examined morphologically. Many morphological characters used to define these taxa are quantitative and overlap substantially among some forms--it is only through suites of such characters that species can be identified. We apply Population Aggregation Analysis and tree building methods to five rapidly evolving gene regions--the mitochondrial AT rich region and the nuclear acetylcholinesterase, hunchback, mastermind, and vestigial loci to test the morphological species delineations within the morphocryptic mayaguana triad. We find that D. mayaguana is diagnosable using DNA sequences, but the other two species form a non-diagnosable paraphyletic assemblage. A single ecological factor, oviposition substrate, is an important diagnostic character distinguishing D. straubae from D. parisiena, highlighting the importance of examining a diverse array of data (morphological, molecular, ecological, and behavioral) when defining species limits.