Understanding the origin and spread of invasive species is critical for predicting when and where new introductions will establish, and impact native species. However, due to the complexity of contributing factors such as multiple introductions, dispersal method, genetic admixture in founding populations, and variable propagule pressure, genetic patterns observed in invasive species may not always conform to a single theoretical expectation. Cuban treefrogs (Osteopilus septentrionalis) are invasive in peninsular Florida and sporadically in the Florida panhandle. Though O. septentrionalis has been occasionally reported in Louisiana since the 1990s, established populations were not present until the discovery of a breeding population in New Orleans in 2017. In this study we investigated the source of this novel population using existing and newly generated cytochrome B (cyt-b) mitochondrial gene sequences from the native and invasive range of O. septentrionalis. We recovered a total of 14 cyt-b haplotypes, nine novel and five previously published. Within the 95 Louisiana invasion samples, we recovered seven haplotypes including five novel haplotypes. The haplotypes most common in Louisiana were shared exclusively with west and east Florida localities in central Florida, indicating a possible source population. The presence of haplotypes private to the Louisiana locality suggests other unsampled localities may also be contributing to the Louisiana settlement. Metrics of genetic diversity across native and invasive localities did not significantly differ. Furthermore, the Louisiana samples had higher genetic diversity than any single location sampled within Florida. Thus, genetic diversity and our haplotype connectivity suggest the Louisiana population is derived from multiple introductions from Florida. Our study highlights how demographic and genetic analyses can be utilized to understand the source and future expansion potential of invasive populations.