Adaptation of pest species to laboratory conditions and selection for resistance to toxins in the laboratory are expected to cause inbreeding and genetic bottlenecks that reduce genetic variation. Heliothis virescens, a major cotton pest, has been colonized in the laboratory many times, and a few laboratory colonies have been selected for Bt resistance. We developed 350 bp Double-Digest Restriction-site Associated DNA-sequencing (ddRAD-seq) molecular markers to examine and compare changes in genetic variation associated with laboratory adaptation, artificial selection, and inbreeding in this non-model insect species. We found that allelic and nucleotide diversity declined dramatically in laboratory-reared H. virescens as compared with field-collected populations. The declines were primarily due to the loss of low frequency alleles present in field-collected H. virescens. A further, albeit modest decline in genetic diversity was observed in a Bt-selected population. The greatest decline was seen in H. virescens that were sib-mated for 10 generations, where more than 80% of loci were fixed for a single allele. To determine which regions of the genome were resistant to fixation in our sib-mated and Bt-selected lines, we generated a dense intraspecific linkage map containing 3 PCR-based, and 659 ddRAD-seq markers. Markers that retained polymorphism were observed in small clusters spread over multiple linkage groups. These markers are likely associated with genomic regions under balancing selection, thus preventing fixation of deleterious alleles.