Second generation anticoagulant rodenticides (SGARs), developed first to exterminate rodent populations, have now become a means of accidental secondary wildlife intoxication. Utilizing highly potent bait formulations designed to overcome resistance, these SGARs now exhibit ongoing persistence in liver and other tissues. Particularly, SGARs persist antemortem within target consumer tissues allowing for non-target wildlife exposure. The aim of this research was to utilize bromadiolone’s and brodifacoum’s diastereomer configurations of cis and trans isomers to determine whether one isomer had less tissue persistence. To accomplish this, High Performance Liquid Chromatography paired with Mass Spectrometry (HPLC-MS/MS) was applied. Through partnership with the California Department of Fish and Wildlife, 45 non-target wildlife livers including coyote (Canis latrans), gray fox (Urocyon cinereoargenteus), and cougar (Puma concolor) were obtained and analyzed for diastereomer proportions of bromadiolone and brodifacoum. Current bromadiolone and brodifacoum baits were tested and isomer ratios were compared to ratios in primary consumer species and secondarily exposed non-target species. All 45 non-target samples contained brodifacoum and 41 had bromadiolone residues. For bromadiolone, trans-bromadiolone was shown to be more persistent in the food chain with 56% (23/41) having ≥91%. For brodifacoum, 35/45 cougar and coyote samples displayed the cis isomer as more persistent. However, gray foxes displayed the trans isomer of brodifacoum. Thus, brodifacoum’s isomer pattern is variable, perhaps reflecting species differences in brodifacoum metabolism and elimination. Moreover, the benefits of reformulating brodifacoum to a certain isomer were not as clear. However, this research establishes that reformulation of bromadiolone bait to include less trans isomer will likely lower both food chain persistency and non-target wildlife exposure.