Napier grass (Pennisetum purpureum Schum.) is a well-established perennial fodder crop of African origin which recently has also drawn attention for its potential as biofuel feedstock. The absence of genome information in Napier grass limits the development of sequence-specific markers which often involves a high developmental cost. This study aimed to determine cross-species transferability of microsatellite markers between pearl millet (Pennisetum glaucum L.) and Napier grass and to assess the genetic diversity of Napier grass accessions. A total of 107 pearl millet microsatellite markers were tested of which 71 markers (66 %) showed successful cross-amplification. Only 29 markers were selected to study the genetic diversity of Napier grass accessions maintained at the US Department of Agriculture-Agriculture Research Service (USDA-ARS) Tifton, GA. A total of 108 alleles were identified among 99 accessions, a pearl millet line, and a sugarcane hybrid. The average polymorphic information content (PIC) value was 0.212 per marker, while Dice coefficient of similarity ranged from 0.50 to 1.0, indicating high genetic variability among accessions. The accessions with the lowest Dice coefficient of similarity values could be useful in breeding programs. The similarity coefficient of accessions equal to 1.00 is likely an indication of a single genotype being represented by two accessions with different names. This study provides an expanded set of microsatellite markers transferable from pearl millet to Napier grass that can be used to evaluate genetic diversity in Napier grass accessions.