Sweet sorghum (Sorghum bicolor L.) has been recognised widely as a potential alternative source of biofuel because of its high fermentable sugar content in the stalk. With international focus on alternative fuel sources, investors are looking at sweet sorghum as a feedstock source for bioethanol production without compromising sorghum’s primary use for food, fodder, and animal feed. However, for researchers to maximize its feedstock potential and to be used in future there is a need to understand the genetic diversity and relationships among sweet sorghum landraces existing in Tanzania. The objective of this study was to assess the genetic diversity and relationships among sweet sorghum accessions using SSR markers, to examine brix level variability within accessions and to establish their genetic relationships for the informed use of these accessions in breeding programs. Forty three accessions [32 accessions from farmers’ fields, 10 accessions from ICRISAT] were genotyped with 42 SSR markers that generated 220 alleles with an average of 5.07 alleles per locus. Polymorphic SSRs revealed a genetic diversity of 0.48 with a range of 0.07 to 0.87 and a total of 220 alleles. Cluster analysis using dissimilarity matrices and hierarchical clustering with un-weighted pair group method with arithmetic averages was employed to analyse the genetic relationships among accessions, thus 43 accessions were grouped into 3 distinct clusters. A sufficiently large degree of genetic diversity was observed in the collected germplasm and ICRISAT Materials. (>0.75 dissimilarity between ICSV_93046 and WEG_Bu; between ICSV_93046 and NJG and between S_Ind_35 and KULY_Mch). However, further evaluation tagging gene for sugar content is important. The information obtained from this study coupled with phenotypic characterization can be used by plant breeders to select parents for improving the sugar content in the adapted sorghum varieties and to develop segregating populations to map genes controlling sugar content in sweet sorghum.