Technical Abstract: The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species that is destructive to crops grown by subsistence farmers in tropical regions of West Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 were shown to have orthologs within the Bombyx mori gene model. A homology-based analysis initially resulted in assignment of M. vitrata genes into group of paralogs, but were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy-one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa, and results indicated that population substructure may be present, which has important implications for managing this insect’s resistance to transgenic cowpea that express Bacillus thuringiensis crystalline toxins. This study shows that mutation data derived from transcriptome sequencing data is an expeditious and economical source for genetic marker development that allows evaluation of ecological differentiation.