Watermelon [Citrullus lanatus (Thunb.) Matsum and Nakai] is one of the economically most important fruit crops of the Cucurbitaceae family. Among different watermelon traits, disease resistance and fruit quality are the important traits for growers and consumers. The single nucleotide polymorphism (SNP) markers similar to those traits can potentially and cost-effectively distinguish the genetic variations among these traits. Consequently, we developed 33 SNP makers linked to different watermelon traits associated with fruit quality and disease resistance, and validated in the genetic resources of watermelon and F1 breeding lines using ‘Fluidigm SNP Genotyping’ assay. Most of the SNP markers distinguished the alleles into three different types such as reference allele, alternative allele and heterozygous from watermelon genotypes for various traits. The SNP markers ‘ZymFL-T81P’ (ZYMV- resistance), ‘FON1-U161’ and ‘FON1-S075’ (Fusarium wilt-resistance), ‘Pmr21-Cla831’ (PM-resistance), and ‘ClGBS-J168’ and ‘GBS-GC230’ (GSB-resistance) can successfully differentiate resistant (R), susceptible (S) and heterozygous watermelon genotypes. Similarly, the SNP marker associated with sugar content, citrulline content, arginine content, rind hardness, flesh firmness, fruit shape, rind strip pattern of watermelon fruit and seed coat colour can successfully distinguished the watermelon genetic resources and F1 breeding lines as reference allele (A) type, alternative allele (B) type and heterozygous (H). These SNP markers could be utilized for marker assisted selection as well as screening of a large number of watermelon germplasm for fruit quality and disease resistance. However, further validation like artificial inoculation of pathogens for the traits related to disease resistance is required in watermelon crops.