Trinidad offers a unique study system within the Caribbean to assess the processes and patterns of amphibian speciation. We used mitochondrial DNA sequences to investigate the phylogenetic relationships and patterns of intraspecific genetic variation of Mannophryne trinitatis from Trinidad. Molecular clock estimates point to a genetic split between M. trinitatis and its sister species, M. venezuelensis, dating to the Late Miocene (c. 7–8 Mya), suggesting vicariance as a means of speciation when Trinidad pulled apart from northern Venezuela. M. trinitatis phylogenetic population analyses from ten Northern Range and four Central Range localities recovered three well-resolved clades: a larger clade formed by haplotypes from Northern Range localities and two additional clades, one formed by haplotypes from the Central Range and another including haplotypes from Northern Range localities and one haplotype from the Central Range. Overall, our results show that the genetic diversity in M. trinitatis is not geographically structured but it is distributed among the various Northern and Central Range localities. In congruence with the vicariance speciation hypothesis, we attribute M. trinitatis present distribution and lack of genetic structure to multiple admixture events caused by climate changes that severely affected the topology of Trinidad throughout the Pliocene ⁄ Pleistocene periods.