This study aimed to broaden applicability of KASP for Oryza sativa across diverse genotypes through incorporation of ambiguous (degenerate) bases into their primer designs and to validate 4,000 of them for genotyping applications. A bioinformatics pipeline was used to compare 129 rice genomes from 89 countries with the indica reference genome R498 and generate ∼1.6 million KASP designs for the more common variants between R498 and the other genomes. Of the designs, 98,238 were for predicted functional markers. Up to 5 KASP each for 1,024 breeder-selected loci were assayed in a panel of 178 diverse rice varieties, generating 3,366 validated KASP. The 84% success rate was within the normal range for KASP demonstrating that the ambiguous bases do not compromise efficacy. The 3,366-trait-specific marker panel was applied for population structure analysis in the diversity panel and resolved them into 4 expected groups. Target variations in 13 genomes used for designs were compared with the corresponding KASP genotypes in different accessions of the same 13 varieties in the diversity panel. There was agreement for 79% or more markers in 12 varieties; 10 having agreement >88%. One variety, a selection from a landrace, had only 46.5% marker agreement. Breeders can search for the validated KASP and more than a million so-far untested designs in three reference genomes (including Niponbare MSU7) with a search tool, that includes designs in proximity to previously published microsatellite markers, and retrieve target variations for 129 rice genomes plus their genomic locations with ±25 bp flanking sequences.