Two large-effect QTLs and 14 candidate genes conferring resistance to Marssonina leaf spot disease were identified in an F1Populus deltoides pedigree, which provided valuable information for cloning the particular underlying genes in future. Marssonina leaf spot disease (MLSD), which is caused by Marssonina brunnea, is a devastating threat to poplar plantations. To map quantitative trait loci (QTLs) underlying resistance to MLSD, an F1P. deltoides pedigree has been established and genetic maps were constructed for the mapping parents according to the two-way pseudo-testcross mapping strategy. The female map contained 913 markers spanning a total genetic distance of 3132 cM, with linkage groups (LGs) corresponding to the 19 haploid chromosomes in poplar, whereas the paternal map contained 252 markers distributed on 22 LGs and covered a genetic length of 1809 cM. The established maps were further aligned to the poplar consensus genetic map based on the integrated SSR markers. The resistance to MLSD was recorded as a complex binary trait based on the black spot symptom on leaves. Analyses of QTLs revealed two large-effect QTLs in LGs VI and XVI, namely qMLSD-VI-1 and qMLSD-XVI-2, which explained 50.3% and 34.5% of the total phenotypic variance, respectively. A significant interaction between these two QTLs was detected based on a two-way ANOVA. In this mapping pedigree, the female parent contributed all of the QTL alleles conferring resistance to M. brunnea. Genome sequences in the target regions were obtained by aligning the QTL intervals to the poplar genome sequence with the mapped SSR markers. The importance and utility of the 14 candidate genes associated with disease resistance identified in the QTL intervals should be more thoroughly characterized in future studies.