Urbanization in wildlife often occurs as a few individuals from rural environments migrate to an urban setting. Within burrowing owls (Athene cunicularia), urbanization has led to numerous consequences including the reduction of fear responses to human presence and the loss of rare alleles. Recent genomic analyses of burrowing owls have also implicated selection on various neuronal and synaptic genes in response to urbanization, linking the transition to urban environments to changes in cognitive function. However, genomic analyses are often computationally intensive, require samples with high quality DNA, and omit a large proportion of DNA sequencing data that cannot be accurately assigned to the draft reference genome. Here, we utilize this proportion of unused data and associate it with environmental variables to assign individual burrowing owls to either urban or rural origin. Our goal is to develop a tool for characterizing the DNA from burrowing owls using only a few short target sequences as predictors. This work will also aid in identifying previously unknown sequences associated with urban and rural environments. Additionally, knowledge of these sequences will allow for the rapid characterization of burrowing owls sampled across the urban-rural landscape to improve our understanding of their range, dispersal, and the effects of urbanization for conservation efforts.