Brook Trout Salvelinus fontinalis in the southern Appalachian portion of their range have been isolated in remote headwater systems for millennia. Recent genetic investigations indicate extremely low allelic diversity, heterozygosity and effective population sizes in many streams. In populations restored using multiple source stocks, limited introgression has been observed despite source stocks being collected from streams within the same subwatershed. It remains unclear if pre- and/or post-reproductive isolating mechanisms are restricting effective gene flow among source stocks in restored streams. Objectives of this study were to: 1) identify environmental variables contributing to assortative mating, and 2) use common garden crossings to determine if wild type brood stock crossings resulted in physiologically viable offspring. We observed markedly different fertilization success rates within-population (66.7%) and betweenpopulation (91.7%) from the 42 crosses (N=18 control, N=24 treatment). Moreover, we observed significant (P < 0.05) differences between within-population and between-population groups in each of our linear mixed effects global models for each trial stage of development (i.e., fertilization rate, eyed egg rate, and hatch rates). Tukey’s HSD comparisons revealed only one significantly (P < 0.003) different fertilization rate among the forty five pairwise comparisons in each of our three stages of trails. In addition, we observed differential peaks of gamete production within and among source stream brood stock, despite common garden conditions, that appeared to have limited fertilization success rates between interstream and control groups. Despite differential peak gamete timing, intrastream crosses performed equally, and, in some instances, better than those between control groups. Our results suggest differential responses to shared environmental conditions (i.e., temperature and/or photoperiod) may contribute to mismatched spawning phenology (i.e., gamete production timing) among restoration founder stocks leading to introgression (i.e., genetic admixture). The application of contemporary genetic techniques could help determine if these possible local adaptations are genetically fixed or may break down over time in restored populations with mixed source stocks. These findings demonstrate the need to apply contemporary conservation genetics tools to future wild trout restoration projects using translocated source stock towards the goal of “genetically-robust”, naturally reproducing populations with the ability to cope with current and future perturbations.