In marker assisted breeding programs, determination of genome polymorphism and development of suitable molecular markers is of the greatest importance. The aim of this research was development of RAPD markers, which will enable quick and cost efficient DNA polymorphism analysis among closely related sugar beet genotypes. The research was conducted on twelve sugar beet genotypes from population of closely related genotypes. Reactions with eight RAPD primers and five primer mixtures resulted in stable and reproducible bands in all samples, with 44 polymorphic and 14 monomorphic loci, and average of 6.13 bands per primer. In two-primer reactions nine new polymorphic bands were detected. Polymorphism information content (PIC) for each primer was calculated, while genetic variation was estimated by calculation of the number of polymorphic loci and their percentage, observed number of alleles, effective number of alleles, and Nei’s gene diversity. An unweighted pair group arithmetic mean method (UPGMA) cluster analysis showed that samples were divided in two groups with relatively high coefficient of similarity. The presented results showed that RAPD markers can be suitable for genetic diversity analysis in breeding material with high levels of homology and homozygosity