Amphibians of the family Bufonidae in Puerto Rico include both endemic species of high conservation concern and widely established invasive species. This study proposes the molecular identification and analysis of genetic variability in the endemic Puerto Rican Crested Toad (Peltophryne lemur) and the invasive Cane Toad (Rhinella marina) using mitochondrial COI DNA barcoding obtained through non-invasive sampling methods. Results will provide genetic references to compare native and invasive populations and support conservation and management strategies for amphibians in Puerto Rico.