Ratoon stunting disease (RSD) is a pervasive bacterial disease in the world, which seriously affects the yield and quality of sugarcane. To clarify the incidence of RSD in various ecological zones, different varieties, and crop cycles in Yunnan, 214 samples were collected from three planting regions situated in the North Tropic Humid and South Subtropical Humid zones. These samples underwent molecular detection for RSD. The results showed that the highest positive detection rate of RSD was 88.57% in Longzhang in the South Subtropical Humid zones. RSD was detected in 18 main planting or promoting varieties, the positive detection rate was 42.86%-100%. For the crop cycles, RSD occurred in different degrees from newly planted to fourth ratoon crop. The results reveal significant differences in RSD incidence dynamics among sugarcane varieties, crop cycles, and ecological areas. Notably, during the transition from newly planted to second ratoon crop, RSD incidence increased with the duration of ratoon years in different ecological sugarcane areas. Furthermore, there was a tendency for RSD incidence to increase with successive ratoon years in varieties Yuetang 93-159, Yunzhe 08-1609, Liucheng 05-136, Yunzhe 05-51, Liucheng 03-182, Chuantang 79-15 and Yunzhe 05-49.