The advantages of target region amplification polymorphism (TRAP) for identification of polymorphisms in coding regions obtained from EST library were used to determine the genetic variability and relationships among 10 sugarcane varieties. TRAP fixed primers based on CDPK and Aqua candidate genes involved in stress tolerance were paired with three arbitrary primers to produce 6 primer combinations.A total of 60 DNA fragments with an average of 10 bands per primer combination were generated from 10 sugarcane genotypes. The percentage of polymorphism was 55.17% and 54.84% in CDPK and Aqua loci, respectively. The largest number of amplified fragments was obtained with the Aqua + arbit.3 primer combination (14 bands), and the lowest (6 bands) with the Aqua + arbit.1. The highest number of TRAP markers was amplified from EI 24-3 OR 2 and G95-19, while the least number of bands was obtained from G.98-28, N.CO.310 and EH 16-9. The lowest similarity were obtained between N.CO.310 and G95-21 reflecting high diversity among them and suggested that crosses between these two genotypes would probably result in the highest variability for stress tolerance. The dendrogram classified the genotypes based on their pedigree in which varieties that have common parent were grouped together based on the polymorphism in CDPK and Aqua loci.The results indicated that the TRAP markers can provide a powerful technique for genetic diversity in sugarcane. The performance of the sugarcane varieties in stalk traits (height, diameter and number) and cane yield were studied in two seasons. Maximum genetic advance along with high heritability was observed for stalk height, reflecting the preponderance of additive gene action in determining such character, so reliable selection could be made for this trait on the basis of phenotypic expression. The other traits displayed high amount of heritability associated with low genetic advance indicating that non-additive gene action governing these traits, which could be improved through hybridization and hybrid vigor.