The goal of this project was to primarily assess east-west connectivity across Route 67 and secondarily, north-south connectivity across Scripps Poway Parkway and Poway Road, two highly trafficked roads to the west of Route 67.The Southern Mule Deer is a mobile but non-migratory large mammal found throughout southern California and is a covered species in the San Diego Multi-Species Conservation Plan. USGS researchers assessed deer movement and population connectivity across California State Route 67 and two smaller roads in eastern San Diego County using non-invasive genetic sampling. They collected deer scat pellets between April and November 2015, and genotyped pellets at 15 microsatellites and a sex determination marker. They successfully genotyped 71 unique individuals from throughout the study area and detected nine recapture events. Recaptures were generally found close to original capture locations (within 1.5 km). They did not detect recaptures across roads; however, pedigree analysis detected 21 first order relative pairs, of which approximately 20% were found across State Route 67. Exact tests comparing allele frequencies between groups of individuals in pre-defined geographic clusters detected significant genetic differentiation across State Route 67. In contrast, the assignment-based algorithm of STRUCTURE supported a single genetic cluster across the study area. Their data suggest that State Route 67 may reduce, but does not preclude, movement and gene flow of Southern Mule Deer.