The genetic improvement (GI) plays an important role in yield increase in rice in the Yangtze River Basin of China, and rice roots might have a crucial function during the GI. However, little is known what changes in root physiology and morphology were during the GI in this area and how they were related with grain yield. In this study, 24 typical mid-season rice cultivars (including hybrid combinations) applied in the area during the last 60 years were grown in the field, and they were classified into six types according to their application decades in which the cultivars were predominantly used. Changes in morphological and physiological traits of roots and their relationships with grain yield during the GI were investigated. Results showed that grain yield was significantly increased. Increase in grain yield was attributed mainly to the increase in total number of spikelets, which resulted mainly from a large panicle. The root dry weight, root length, root diameter, root oxidation activity, total absorbing surface area and active absorbing surface area of roots were also increased with the GI. Regression analysis showed that the root dry weight, root length, root diameter, root oxidation activity, total absorbing surface area and active absorbing surface area of roots were very significantly and positively correlated with grain yield. The results suggest that the GI improves root morph-physiological traits which benefits shoot growth, leading to a higher grain yield for the modern cultivars, especially for super rice cultivars.