The rich genetic diversity inCitrullus lanatusand the other six species in theCitrullusgenus provides important sources in watermelon breeding. Here, we present theCitrullusgenus pan-genome based on the 400Citrullusgenus resequencing data, showing that 477 Mb contigs and 6249 protein-coding genes were absent in theCitrullus lanatusreference genome.In theCitrullusgenus pan-genome, there are a total of 8795 (30.5%) genes that exhibit presence/absence variations (PAVs). Presence/absence variation (PAV) analysis showed that a lot of gene PAV were selected during the domestication and improvement, such as 53 favorable genes and 40 unfavorable genes were identified during theC. mucosospermustoC. lanatuslandrace domestication. We also identified 661 resistance gene analogs (RGAs) in theCitrullusgenus pan-genome, which contains 90 RGAs (89 variable and 1 core gene) located on the pangenome additional contigs. By gene PAV-based GWAS, 8 gene presence/absence variations were found associated with flesh color. Finally, based on the results of gene PAV selection analysis between watermelon populations with different fruit colors, we identified four non-reference candidate genes associated with carotenoid accumulation, which had a significantly higher frequency in the white flesh. These results will provide an important source for watermelon breeding.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09443-w.