Bitterness, which is mainly attributed to limonoids, seriously affects the taste of pomelo fruits and hinders their competitiveness in the market. Therefore, a high-performance liquid chromatography method was developed for simultaneous isolation and determination of limonin, nomilin, and limonin glucoside contents in pomelo fruit, and the method showed good precision (RSD ≤ 2.85 %), repeatability (RSD ≤ 3.37 %) and high sample recovery rate (95.8 %–99.7 %). Four pomelo varieties were used to investigate the accumulation and distribution of limonoids in different stages of fruit development and in different plant tissues and varieties, as well as to reveal the synthesis mechanism and provide a basis for the utilisation of pomelo resources in different industries. Results showed that flavedo was the tissue richest in limonoids, and the maximum limonoid content could reach 5959 μg/g fresh weight. During fruit growth and development, the limonin and nomilin contents in juice sacs and albedo first increased and then decreased. Although there were some fluctuations, the limonin and nomilin contents in the flavedo generally showed a decreasing trend with fruit development. Our investigation of the LGT gene expression pattern provided the basis for exploring natural debittering mechanisms in pomelo. Expression of the LGT gene was positively correlated with limonin glucoside content.