Mixed farming of rice and millet is one of the basic agricultural modes in the upper and middle Huai River Valley(HRV). According to the latest data, this agricultural mode appeared during the middle and late Peiligang Culture(7.8–7.0 ka BP) in the upper HRV, and then became a common subsistence economy in the end of the Neolithic(5.0–4.0 ka BP) in both the upper and middle HRV. However, it is still not clear how this mixed farming developed in the upper HRV after its occurrence, nor are the regional differences in the development of mixed farming between the upper and middle HRV during the Neolithic completely understood. In this paper, flotation and starch analyses were conducted on samples from eight archaeological sites in the upper and middle HRV. The results indicate that the mixed farming of rice and millet first appeared in the later phase of the middle Neolithic in the regions of the Peiligang Culture, then developed quite rapidly in the late Neolithic(6.8–5.0 ka BP), finally becoming the main subsistence economy at the end of the Neolithic in the upper HRV. However, there are obvious differences in the emergence and development of agriculture between the middle and upper HRV. Rice farming was the only agricultural system during the middle Neolithic, lasting until the end of the Neolithic, when mixed farming appeared in the middle HRV. Furthermore, although mixed farming appeared in both the upper and middle HRV during the end of the Neolithic, the roles of rice, foxtail millet and broomcorn millet in the subsistence economy were not the same. In general, millet was more widely cultivated than rice in the upper HRV, but rice occupied the same or a slightly more prominent position in the middle HRV at the end of the Neolithic. These results are significant for understanding the process of agricultural development and transformation, as well as human adaptation to climatic and cultural variability duringthe Neolithic. Mixed farming of rice and millet is one of the basic agricultural modes in the upper and middle Huai River Valley (HRV). According to the latest data, this agricultural mode was during the middle and late Peiligang Culture (7.8-7.0 ka BP) in the upper HRV, and then became a common subsistence economy in the end of the Neolithic (5.0-4.0 ka BP) in both the upper and middle HRV. However, it is still not clear how this mixed farming developed in the upper HRV after its occurrence, nor are the regional differences in the development of mixed farming between the upper and middle HRV during the Neolithic completely understood. In this paper, flotation and starch analyzes were conducted on samples from eight archaeological sites in the upper and middle HRV. indicate that the mixed farming of rice and millet first appeared in the later phase of the middle Neolithic in the regions of the Peiligang Culture, then developed quite rapidly in the late Neolithic (6.8-5.0 ka BP), fi nally becoming the main subsistence economy at the end of the Neolithic in the upper HRV. However, there are obvious differences in the emergence and development of agriculture between the middle and upper HRV. Rice farming was the only agricultural system during the middle Neolithic, lasting until the end of the Neolithic, when mixed farming appeared in the middle of the Neolithic, when mixed farming appeared in the middle of the Neolithic, the mixed roles of the millet in the subsistence economy were not the same. In general, millet was more widely cultivated than rice in the upper HRV, but rice occupied the same or a slightly more prominent position in the middle HRV at the end of the Neolithic. These results are significant for understanding the process of agricultural development and transformation, as well as human adaptation to climatic and cultural variability during the Neolithic.