Abstract Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical difficulties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a modified CTAB DNA isolation protocol referred to as the CTAB-LiCl DNA extraction method was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3–79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as confirmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisulfite sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or beta-mercaptoethanol and can be completed in approximately 2 h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 µg to 28 µg without affecting the seeds’ germination ability, allowing marker-assisted selection before planting and flowering.