Genetic diversity among 101 cottons varieties, including 72 cultivars from the Huanghe valley and 29 cultivars from the Changjiang valley, was investigated using AFLP markers. In total, 20 primer combinations revealed 200 polymorphic bands among the Huanghe valley cottons and 127 polymorphic bands among the Changjiang valley, respectively. Euclidean distance values were calculated using SPSS (11.0) software. Average Euclidean distance value was 4.356 (the Huanghe valley) and 4.391 (the Changjiang valley), respectively. When the value was 15.2, 72 varieties from the Huanghe valley were divided into four groups (the Huanghe valley groups, abbrevate HVGs), including HVG1 (27), HVG2 (19), HVG3 (10) and HVG4 (16). 29 varieties from the Changjiang valley were classified into four groups (the Changjiang valley groups, abbreviate CVGs), including CVG1 (14), CVG2 (4), CVG3 (5) and CVG4 (6). Compared with Euclidean distance matrices and frequency distribution of pairwise Euclidean distances of cottons from the Huanghe and Changjiang valleys, it was showed that there was similar genetic diversity between the cotton cultivars from Huanghe valley and those from Changjiang valley.