Hybridization is an important evolutionary process that contributes to the creation of novel genetic recombinants where species interbreed. Following recombination, differential introgression of bi-parentally and uni-parentally inherited genomes can provide signatures of the extent and direction of gene flow. Assessment of biparental and uniparental genome introgression is key to understanding cytonuclear interactions following secondary contact. Indeed, comparison of nuclear and chloroplast introgression can provide a test for incongruencies that may arise during hybridization. In this study, we leverage whole genome resequencing data from 576 Populus trees across seven latitudinally distributed transects spanning the natural contact zone between Populus trichocarpa and P. balsamifera. We used replicated hybrid zones to test for differential introgression between nuclear and chloroplast genomes across environments. Populus provides an excellent model to evaluate differential patterns of introgression due to its weak barriers to reproduction that lead to extensive hybridization in nature. Furthermore, small genome size, ease of clonal propagation, and importance as a source of lignocellulose, make Populus an important economic model to evaluate the fitness consequences of cytonuclear interactions. We assembled the chloroplast genome for 576 Poplar genomes using NOVOPlasty. Following this, we identified the probability of chloroplast genome ancestry using maximum likelihood phylogenetic trees. We then compared nuclear genomic ancestry with chloroplast genome ancestry to test for potential cytonuclear discrepancies across replicate hybrid zones. Finally, Ka/Ks ratios were used to test for signatures of selection in the chloroplast genome across the replicated hybrid zones. Overall, the chloroplast genome of both species is highly conserved with genome sizes varying from 156,235 to 157,980 bp, includes 77 coding regions, and the typical quadripartite structure (LSC, SSC, IRA, and IRB) of angiosperms. Across the southern transects, we observed a transition from the P. trichocarpa chlorotype to the P. balsamifera chlorotype suggesting the maintenance of both haplotypes across the hybrid zone. However, in the northern transects, the P. balsamifera chlorotype was the sole chlorotype present. Comparisons between nuclear and chloroplast introgression suggest that unidirectional gene flow of P. balsamifera chlorotypes into a P. trichocarpa nuclear genomic background may persist across the hybrid zone. Tests for selection indicate that some chloroplast genes exhibit more than one non-synonymous substitution, but the distributions of non-synonymous substitutions vary geographically across latitudinal transects. Future work will evaluate how cytonuclear interactions influence phenotypic variation using common garden experiments.