Genetic diversity and fingerprint of varieties are crucial reference for breeding, cultivar right protection and new cultivar extension. We used nine pairs of AFLP markers and 15 pairs of SSR markers to scan 38 new sugarcane varieties (lines) from the national variety regional testings, obtaining abundant fingerprint data. Among the total 348 AFLP bands, 248 were polymorphic, with a polymorphism rate of 72.26 %. In addition, among the total 180 SSR bands, 176 were polymorphic, with a polymorphism rate of 97.78 %. The genetic similarity coefficients of these 38 new varieties (lines) were distributed from 0.668 to 0.847. We explored the distribution characteristics of genetic similarity coefficients using the boxplot, observing the genetic basis of five series (FN, MT, YZ, YG, and GT) of the 38 new sugarcane varieties (lines) was approximately similar. The clustering analysis manifested that these 38 new sugarcane varieties (lines) were divided into two groups at the genetic similarity coefficient of 0.732, with a subgroup including FN09-2201 and GT06-1492 that had high heterogeneity. Moreover, there was a small subgroup containing ROC22 at the genetic similarity coefficient of 0.770. Except for ROC22, the subgroup also contained FN 07-3206, FN 40, HZ 22, GT 09-12, and LC 07-150. ROC22 has wide adaptability, high yield, high sugar content and other excellent characteristics, other varieties (lines) in the same subgroup should be more likely to have these excellent characteristics and higher extention potential. Finally, through selection of 60 efficient amplification sites in the identification of SSR markers, we constructed the fingerprints of these 38 new sugarcane varieties (lines), which should play an important role in variety identification and variety protection. This study is expected to be directly applied to guide the genetic diversity assessment and molecular fingerprinting identification of sugarcane germplasm, and also to provide references for the extention and layout of these varieties or their utilization as hybrid parents.