Cotton (Gossypium spp.) is one of the most important economic crops in the world. It not only produces natural fiber for textile industry, but also provides a large quantity of cottonseeds enriched with high-quality protein and oil. However, the presence of gossypol limits the utilization of the cottonseeds. Two pairs of cotton near isogenic lines (NILs) with different types of pigment glands, CRI12 and CRI12W and Coker 312 and Coker 312W, exhibit different gossypol contents in plants. The glandless traits of CRI12W and Coker 312W are mainly controlled by dominant and recessive genes. However, less is known about genomic differences in NILs. In the current study, next-generation sequencing was used to discover the relationship between phenotypes and DNA polymorphisms in these NILs. The whole genomes of CRI12, CRI12W, Coker 312 and Coker 312W were resequenced. The sequencing depths were 34.01, 37.60, 36.27 and 35.88, respectively. Genomic variations of the NILs were identified in comparison with the reference genome of TM-1. A total of 2371614, 2045733, 2013021 and 1974262 SNPs, 181706, 180714, 177324 and 167971 Indels, 4208, 4026, 3963 and 3771 SVs, and 46741, 35976, 36388 and 34765 CNVs for CRI12, CRI12W, Coker 312 and Coker 312W were uncovered, respectively. Gene Ontology (GO) analysis of the genes with differential SNPs and Indels in two NILs revealed that variations were enriched in different ontology terms. KEGG pathway analysis figured out genes with differential SNPs and Indels were mainly enriched in biosynthesis of secondary metabolites pathway and sesquiterpenoid and triterpenoid biosynthesis pathway. Quantitative RT-PCR (qRT-PCR) analysis revealed that key genes with variations participating in the pathway of gossypol biosynthesis and pigment glands formation had a different expression pattern in two NILs. Through next-generation sequencing, a large number of genomic variations were revealed. These DNA polymorphisms provided deeper insight into cotton lines with different types of pigment glands. Further comprehensive analysis revealed that genomic variations were tightly associated with phenotypic difference.