DNA methylation is an important epigenetic modification regulating gene expression, genomic imprinting, transposon silencing and chromatin structure in plants and plays an important role in leaf senescence. However, the DNA methylation pattern during Gossypium hirsutum L. cotyledon senescence is poorly understood. In this study, global DNA methylation patterns were compared between two cotyledon development stages, young (J1) and senescence (J2), using methylated DNA immunoprecipitation (MeDIP-Seq). Methylated cytosine occurred mostly in repeat elements, especially LTR/Gypsy in both J1 and J2. When comparing J1 against J2, there were 1222 down-methylated genes and 623 up-methylated genes. Methylated genes were significantly enriched in carbohydrate metabolism, biosynthesis of other secondary metabolites and amino acid metabolism pathways. The global DNA methylation level decreased from J1 to J2, especially in gene promoters, transcriptional termination regions and regions around CpG islands. We further investigated the expression patterns of 9 DNA methyltransferase-associated genes and 2 DNA demethyltransferase-associated genes from young to senescent cotyledons, which were down-regulated during cotyledon development. In this paper, we first reported that senescent cotton cotyledons exhibited lower DNA methylation levels, primarily due to decreased DNA methyltransferase activity and which also play important role in regulating secondary metabolite process.