Abstract Napier grass is a perennial tropical forage that is used in beef and dairy production systems. Despite its significance in animal nutrition, molecular information available, such as microsatellite or simple sequence repeat (SSR) or single nucleotide polymorphism (SNP) markers, is limited. Using an assembled transcriptome, 50 novel SSR markers were developed, of which 21 were found to be polymorphic. These polymorphic markers were tested for DNA fingerprinting of Embrapa cultivars, five of which revealed distinct allele patterns for cultivar identification. SSR markers 05, 17, and 44 identified a unique pattern in the BRS Kurumi cultivar. The BRS Capiaçu cultivar was identified using SSR markers 17, 43, and 44. The Pioneiro cultivar exhibited a rare fragment amplification pattern using SSR marker 46, while SSR marker 44 revealed a distinct allele in the BRS Canará cultivar. SSR marker panels could be utilized as DNA fingerprinting tools to assist in cultivar identification.