Abstract: 【Objective】 This study aimed to develop KASP （kompetitive allele-specific PCR） molecular markers based on whole-genome resequencing data and perform genetic diversity analysis and DNA fingerprint construction， thereby providing molecular tools and theoretical references for cassava germplasm identification， superior resource exploration， and breeding of new varieties. 【Method】 Based on whole-genome resequencing data of 377 cassava germplasms， highly polymorphic KASP molecular markers were screened according to conditions including locus integrity，minor allele frequency，heterozygosity，polymorphism information content （PIC），GC content，sequence homology，and distribution density of continuous single-base repeats and loci， and genetic diversity analysis and DNA fingerprint construction of 184 cassava germplasm resources were performed. 【Result】 A total of 9443 loci were screened from the 52722 SNP loci at different positions of genome of the whole-genome resequencing data of cassava. A total of 20 SNP loci that were evenly distributed were selected from 18 chromosomes of cassava， which were 360 loci in total. The 22 cassava germplasms were preliminarily screened using 360 pairs of KASP primers，and 27 highly polymorphic KASP molecular markers were identified. Genetic diversity analysis based on the 27 KASP molecular markers in 184 cassava germplasm resources showed that the major allele frequency （MAF） ranged from 0.440 to 0.842，with an average of 0.637； the gene diversity （GD） ranged from 0.270 to 0.591，with an average of 0.464； the GDs of 26 KASP molecular markers were higher than 0.300； observed heterozygosity （Ho） was 0.173-0.538， with an average of 0.344， and 45.3% of KASP molecular markers had Ho distributed in 0.300-0.400； PIC was 0.234-0.545， and 85.18% of KASP molecular markers had PIC higher than 0.300， exhibiting a high level of polymorphism. Cluster analysis grouped the 184 cassava germplasms into 9 major categories，but no clear correlation was observed between the clustering pattern and geographical origin，suggesting extensive gene flow and genetic admixture among cassava germplasms. The genotyping results of 27 KASP molecular markers were transferred to binary coding data for constructing a DNA fingerprint of 184 cassava germplasm resources， effectively revealing genetic differences among different cassava germplasms. 【Conclusion】 The 27 KASP molecular markers are developed based on whole-genome resequencing data of cassava， which can effectively evaluate the genetic diversity and identify genetic relationships among germplasms， therefore， they can be used for germplasm identification， gene localization， and molecular-assisted breeding of cassava.