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Napier grass (Pennisetum purpureumSchum.) is an important forage crop in tropical areas although little is known about its genome information, and few molecular markers have been developed for this species. This work aimed to check the viability of cross‐species amplification of microsatellite markers between pearl millet (Pennisetum glaucum) and Napier grass and to evaluate the genetic diversity among Napier grass germplasm accessions. Fifty‐four microsatellite markers previously described in pearl millet were tested against Napier grass, and 30 markers (55.5%) showed successful cross‐amplification. From them, 18 microsatellite markers were selected to study the genetic diversity in the Embrapa Active Germplasm Bank of Napier Grass (Embrapa‐BAGCE). A total of 180 alleles were identified by these selected microsatellite markers in 107 Napier grass accessions and four pearl millet samples. The average similarity coefficient (Dice) calculated among the Embrapa‐BAGCE accessions was 0.651, ranging from 0.254 to 1.0. Some accessions showed similarity coefficients equal to one, indicating that they have common progenitors or that they might be the same accessions with different denomination. To our knowledge, this work is the first to describe microsatellite markers in Napier grass and represents a significant advance regarding the use of molecular markers in this species.