In this study, 182 wheat cultivars (lines) were tested for leaf rust resistance with mixed races of KHST, FHKT and FHJT of Puccinia triticina at seedling stage. Out of that, 14 cultivars (Taikemai5303, Zhumai305, Yushengmai118, Cunmai18, Xuanmai6, Nongfengchuan, Danmai118, Zhengmai103, Zhengmai119, Saidemai5, Zhengmai369, Xuke918, Yumai668 and AF116-120) were resistant, and the remaining cultivars (lines) were highly infected. By inoculation with 15 Pt pathotypes, which were used for clarifying Lr resistance genes, six cultivars including Zhumai 305, Cunmai 18, Nongfengchuan, Zhengmai 119, Saidemai 5, Zhengmai 369 and Xuke 918 were predicted to the carriers of leaf rust resistance genes Lr33+34. The leaf rust resistance in Zhengmai 103 was possibly caused by leaf rust resistance genes Lr10 and Lr33+34, while that of AF116-120 was possibly derived from leaf rust resistance genes Lr10, Lr16, Lr20 and Lr33+34. These cultivars including Taikemai 5303, Yushengmai118, Danmai 118 and Yumai 668 might contain other Lr genes. In addition, 25 molecular markers for 20 known Lr genes were further deployed for molecular-assisted identification. Lr1 and Lr26 were found to be present in cultivars Nongfengchuan, Xuanmai 6, Zhengmai 103 and Xuke 918, and Lr1 was detected in Taikemai 5303, Shengyumai 118, Zhengmai 119 and Zhengmai 369. Zhumai 305, Cunmai 18 and Yumai 668 carried Lr26; AF116-120 carried Lr1 and Lr2c; Danmai 118 carried Lr26 and Lr37. The results suggested that only few of wheat cultivars were found with Lr genes. Taken together, the results may lay scientistfic basis for the extension and rational distribution of cultivars, leaf rust control and wheat breeding of disease resistance in Huang-Huai-Hai wheat cultivating areas.