Seagrass meadows are rapidly vanishing due to coastal development and climate change. In seagrass research, omics and molecular profiling tools are timely, allowing for new prospects to worldwide concerns. Hence, the present study tested the eight SCoT primers on Cymodocea serrulata and Cymodocea rotundata, which showed 591 polymorphic bands (90.92%), indicating that the individuals are from different populations from the Palk Bay Coast. The Nei-based genetic distance matrix of eight populations of C. serrulata and C. rotundata showed high genetic distance at the intraspecies level in 0.325–0.506. The Nei genetic diversity (h), Shannon information index (I), and allelic number (Ne) of C. serrulata and C. rotundata were low to moderate. C. serrulata had a higher Shannon index (0.54) and genetic diversity (0.36) than the other seven populations, whereas C. rotundata had a higher Shannon index (0.5) and genetic diversity (0.33) in Kattumavadi. The study concludes that the genetic diversity of C. serrulata and C. rotundata shows moderate levels across the study. The Cymodoacea species showed low genetic diversity and heterozygosity due to habitat degradation. These restricted gene flow and fragmented populations are typically exposed to increase their risk of extinction. The population genetics of Cymodoacea species from the Palk Bay region is the first to use SCoT markers to provide insight on seagrass genetics as an ecological indicator for conservation and coastal management.