|Abstract||The Puerto Rican Crested Toad (Peltophryne lemur) is the only native toad in Puerto Rico. It was once found in great abundance along the north and south coastal areas of Puerto Rico. The toads' populations have become severely depleted due to the loss of habitat and the introduction of the marine toad Bufo marinus, to control the sugar cane beetle in the 1920s. Currently, there is a single wild population in Southern Puerto Rico and there are also captive populations in the south and north of Puerto Rico, being maintained by the Fort Worth Zoo (through captive breeding and reintroductions) as a hedge against extinction. Recently, another population in southern Puerto Rico has been discovered with unknown population origins about 3 km away from the original southern population. We extracted DNA from 47 individuals and genotyped them with six microsatellite loci and sequenced the mitochondrial control region to determine the genetic diversity of the population and if it was similar or different from the original southern population. The new population had genetic diversity levels that were similar to the original population, the mitochondrial haplotypes matched the southern population's haplotypes, and the microsatellite allele frequencies were statistically similar to the southern population, indicating little divergence. These data suggest that this new population was the result of a colonization event from the original southern population. Currently listed as critically endangered by the IUCN, our conclusion that this new population was a colonization from the known population gives hope to the species' ability to survive and expand their range.