African Journal of Biotechnology, 2018; 17(26), 811-817Pineapple [Ananas comosus (L.) Merr.] is an important fruit crop cultivated in Tanzania. However, the knowledge on genetic diversity of the pineapple cultivars grown in Tanzania is limited. This study was aimed at determining the genetic diversity and identity of pineapple cultivars from different growing regions in Tanzania using microsatellite markers also known as simple sequence repeat marker (SSR). Ten of the 18 microsatellite markers were polymorphic and generated a total of 22 distinct reproducible bands with an average of 2.2 bands per primer pair. The number of polymorphic bands detected with each primer pair ranged from 1 to 3 with an average of 1.5 per primer pair. The polymorphic information content (PIC) values of each primer pair ranged from 0.17 to 0.79 with an average of 0.41. Two microsatellite loci TsuAC010 and TsuAC039 revealed PIC values higher than 0.50 thus suggesting that such primers have high discriminatory ability. The consensus tree derived from the unweighted pairgroup method with arithmetic means (UPGMA) revealed four different groups. Kinole-SCT subpopulation formed a distinct group from Madeke-SCT and MD2 hybrid cultivar. Kinole-SC, Mukuranga- SC, and Kiwangwa-SCcultivars were closely related on the cluster analysis. This study demonstrated the existence of low genetic diversity in pineapples cultivated in Tanzania implying that a well-thoughtout breeding strategies should be employed for genetic improvements of pineapple. Introduction of exotic clones and employment of modern breeding strategies such as marker assisted selection (MAS) and genetic engineering technologies is recommended. This will widen the current genetic pool of pineapple in Tanzania.