Abstract: Increasingly scarce water resources restrict agricultural development. In the new period, the development of the Yangtze River Economic Belt, the ecological conservation and high quality development of the Yellow River Basin have become major national strategies. Most of the areas where the Yangtze and Yellow rivers flow are important agricultural production areas in China. Their agricultural production must move to a higher quality stage. In this context, the study and comparison of agricultural water use efficiency along the Yangtze River area and the Yellow River area has important reference value for the high-quality agricultural development of the two regions. In the framework of data envelopment analysis, based on panel data of provinces in China from 2000 to 2019, this paper constructs EBM model to measure agricultural water use efficiency according to the measurement idea of “ideal value/actual value of agricultural water”. In addition, multi-level analysis and comparison are made in the two areas from the aspects of spatio-temporal evolution and regional differences. The results show that, firstly, except for Shanghai, efficiency of other provinces in the two areas improve obviously. The efficiency of the area along the Yellow River is obviously lower than that of the area along the Yangtze River before 2016. After 2016, the area along the Yellow River exceeds the Yangtze River. Secondly, although the distribution pattern development of efficiency in the two areas differ greatly in the early sample period, the two areas show high similarity in the later period, with the main peak position moving to the right and the phenomenon of left trailing. Thirdly, the interaction degree within the area along the Yangtze River is low, and its spatial effect is not obvious. The area along the Yellow River shows a “low-low, high-high” agglomeration phenomenon, and has a strong spatial correlation. Fourthly, the regional differences in the two areas show a trend of fluctuation and decline. Ultra-variable density and between-region differences are the main driving forces of the regional differences in the area along the Yangtze River and the Yellow River respectively. Therefore, all provinces in the area along Yangtze River should give full play to their comparative advantages and take targeted measures to improve agricultural water use efficiency. The area along the Yellow River should strengthen coordination and adhere to the road of benign development of “promotion in coordination and coordination in promotion”.