Agricultural development may produce highly fragmented landscapes, which can influence population genetic structure for organisms restricted to insularized breeding habitats. Common Nighthawks (Chordeiles minor) in agriculturally dominated landscapes nest on urban gravel rooftop and might be subject to reduced gene flow among isolated urban sites. We conducted an analysis of the genetic variability and population genetic structure of Common Nighthawks nesting on rooftops in four towns in southeastern South Dakota, an area that has experienced extreme conversion of native prairie to agriculture. Using sequence data from a 725 bp region of the cytochrome b (cytb) mitochondrial gene sampled from 37 individuals representing a total of 30 different nests collected in summers 2014–2016, we found evidence of a recent population bottleneck and reduced mtDNA variability. The spatial dispersion of the 14 detected haplotypes and the presence of nine haplotypes unique to one of the four sampled towns further suggested a lack of panmixia among these nighthawks. These data are consistent with a hypothesis of habitat fragmentation due to agricultural expansion resulting in population isolation within this species of conservation concern.