Rapidly evolving nuclear coding sequences are highly desirable for phylogenetic studies of closely related species. Here, we investigated an 807-bp region, homologous to the testis-specific Tektin gene from Bombyx mori (L.), in 34 nymphalid butterfly taxa in the subfamilies Ithomiinae, Danainae, and Heliconiinae. Within Ithomiinae, relationships inferred from Tektin sequence data were remarkably similar to those in trees based on combined morphological and ecological data. Partitioned Bremer analysis, with mitochondrial cytochrome oxidase I and II, and nuclear wingless and elongation factor 1-α sequences, revealed Tektin to have the greatest utility for inferring relationships at the genus, tribe, and subfamily levels across the studied taxa. We think Tektin will provide a useful source of molecular characters for inference of relationships among other butterflies, and perhaps among other insect taxa.