Maize rough dwarf disease (MRDD) is a severe virus-conferring disease in multiple maize cultivation areas in China. Exploration of resistant germplasm resource and application in resistance breeding is the most economical and effective measure. In this paper, 201 maize germplasm accessions were subjected upon natural inoculation over two years in a high incidence area for MRDD. Out of them, 25 accessions were tested by artificial inoculation. Under field condition, one accession “Shen 137” turned to be highly resistant; 12 inbred lines (P138, Dan 3130, Liao 68, Qi 318, Huangyesi, CA339, H191, Qi 319, X178, SH15, Jinhuang 59 and R18) were resistant; nine accessions (835, Zhongzi 01, 9046, Duohuang 29, CA335, Jinhuang 96B, Hai 9-21, Jinhuang 55 and Shen 136) showed moderate resistance. Notably, the phenotypic datasets gained from two years showed significant different (F = 69.999, P < 0.01). Moreover, by tests for disease resistance by deployment of artificial inoculation, two accessions P138 and R18 which turned to be moderately resistant, whereas Huangyesi and Jinhuang 55 were highly susceptible with disease index of 46.15 and 82.14, respectively. The germplasms showing resistant, except Jinhuang 96B, were selected from the hybrid P78599. Collectively, this study identified several maize inbred lines with rough dwarf disease resistance, valuable in maize breeding for MRDD resistant varieties.