Axillary bud of cotton plants influences cultivation management and mechanical harvesting of cotton. Elucidation of molecular mechanisms of axillary bud development could be helpful in breeding. In this study, the axillary buds during different growth stages of upland cotton cultivar CCRI 9A02 with low-axillary bud phenotype and its mutant plant with high-axillary bud phenotype were analyzed by comparative metabolomics to identify the inherent mechanism for the formation of different axillary bud type. The results showed 56 metabolites were differentially changed between high and low-axillary bud phenotype, and 58.9% of differential metabolites were enriched in early stage, intermediate stage, and growth stage of high-axillary bud phenotype (HP) plant, whereas 41.1% of them were prominently decreased compared with early stage, intermediate stage, and growth stage of low-axillary bud phenotype (LP) plant, respectively. Three common differential metabolites were identified in all three groups of HP vs LP, including N-Acetyl tryptamine (phenolamides), quercetin 5-O-malonylhexosyl-hexoside (flavonol), and chrysoeriol O-acetylhexoside (flavone). Further, with the focus on the flavone and flavonol biosynthesis, and tryptophan metabolism pathway, we revealed that metabolites of flavone hexosides and level of tryptophan metabolism were changed between the development of axillary bud in two cotton cultivars, and the differences observed at three stages suggest that they have different regulation mechanisms, which may be the primary factors leading to the differences of axillary bud among HP and LP cultivars. These results above provide important insights into the axillary bud formation mechanism of upland cotton and will be beneficial for breeding cotton varieties suitable for simplified cultivation.