Assisted gene flow is used to counteract genetic erosion in small populations of endangered species, yet an evaluation of genetic compatibility of potential donor populations and recipient populations is rare. We developed new metrics for assessing genetic impact of genetic augmentation based on genotype identity of functional variants between donor and recipient genomes and used these metrics to evaluate options for assisted gene flow in Eastern Massasauaga rattlesnake (Sistrutus catenatus) populations in Ohio, USA. We used putatively deleterious variants, and genetic variants likely under positive selection (termed “adaptive” variants) as the two components of functional variation. For potential donor and recipient populations, we estimated three key aspects of genetic compatibility – (a) introduction of novel variants, (b) masking or unmasking of existing deleterious variants, and (c) potential for outbreeding depression through disruption of local adaptation. The main impact of augmentation from diverse donor populations was to introduce novel deleterious variants and to a lesser extent novel adaptive variants into each recipient population. Both donor populations had a similar minor impact in terms of masking existing deleterious variants. Finally, only ~ 7% of adaptive variants show evidence for local adaptation arguing that the negative effect of outbreeding depression would be small. These results draw attention to the importance of considering the potential impact of both deleterious and adaptive genetic variants in augmentation efforts and suggest that in case of these endangered rattlesnakes, the net effect of proposed assisted gene flow may lead to an increase in absolute levels of mutation load.