Citrus species among the most important and widely consumed fruit in the world due to Vitamin C, essential oil glands, and flavonoids. Highly variable simple sequence repeats (SSR) markers are one of the most informative and versatile molecular markers used in perennial tree genetic research. SSR survey ofCitrus sinensisandCitrus maximawere identified perfect SSRs spanning nine chromosomes. Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. We designed and validated a class I SSRs in theC. sinensisandC. maximagenome through electronic polymerase chain reaction (ePCR) and found 83.89% inC. sinensisand 78.52% inC. maximaSSRs producing a single amplicon. Then, we selectedextremely variableSSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across seven draft genomes of citrus, which provided us a subset of 84.74% inC. sinensisand 77.53% inC. maximahighly polymorphic SSRs. Out of these, 129 primers were validated on 24 citrus genotypes through wet-lab experiment. We found 127 (98.45%) polymorphic HvSSRs on 24 genotypes. The utility of the developed HvSSRs was demonstrated by analysing genetic diversity of 181 citrus genotypes using 17 HvSSRs spanning nine citrus chromosomes and were divided into 11 main groups through 17 HvSSRs. These chromosome-specific SSRs will serve as a powerful genomic tool used for future QTL mapping, molecular breeding, investigation of population genetic diversity, comparative mapping, and evolutionary studies among citrus and other relative genera/species.