In order to identify new sources of resistance to leaf rust, a globally important disease of wheat, caused by the fungal pathogen Puccinia triticina Eriks. (Pt), a total of 170 hexaploid breeding lines, which have similar morphology with wheat and were derived from crosses of wheat-Thinopyrum ponticum or wheat-Th. intermedium partial amphiploids with susceptible wheat cultivars, were inoculated with the selected Chinese Pt races for evaluating seedling leaf rust response in the greenhouse. The response of adult plants to leaf rust was assessed on seedling susceptible lines over three cropping seasons from 2017 to 2020. In addition, 24 specific markers for 20 known Lr genes were used. Based on the results from gene postulation and molecular marker detection, two adult plant resistance (APR) genes (Lr34 and Lr37) and nine seedling resistance genes (Lr1, Lr2a, Lr2b, Lr3ka, Lr10, Lr15, Lr17, Lr18, and Lr26) were identified in 118 lines either singly or in combination. Most commonly identified Lr genes were Lr37 in 81 (47.6%) lines and Lr26 in 40 (23.5%) lines. Less frequently detected genes were Lr1 in nine lines; Lr2b and Lr3ka each in eight lines; Lr17 in seven lines; Lr2a and Lr34 each in four lines; Lr10 in three lines; Lr18 in two lines; and Lr15 was found in only one line. Twenty-eight lines were resistant to almost all the races used, but no known resistance gene was identified except Lr26, a gene with widespread ineffectiveness, suggesting that these derivatives could possess uncharacterized seedling Lr gene(s). Eleven seedling-susceptible lines were found to have APR over three cropping seasons, with an average maximum disease severity (MDS) below 15%, and hence could be potential sources of new APR genes. This study showed that these hexaploid breeding lines derived from wheat-Thinopyrum amphiploids could serve as new sources for wheat leaf rust resistance.