Alternaria blotch, caused by Alternaria alternata f. sp. mali, is a destructive defoliating disease that affects apple worldwide, especially in Asia. Despite previous efforts in characterizing this disease, few have reported on nucleotide binding sites (NBS)-encoding genes in response to pathogen infection. In this study, we characterized the morphological and gene expression response of ‘Golden Delicious’ and ‘Starking’ apples challenged by A. alternata. Inoculation of detached leaves of ‘Golden Delicious’ resulted in a rapid initiation of disease symptoms but a slow spread of the infection from the point of inoculation. In contrast, detached leaves of ‘Starking’ exhibited a delayed visibility of infection but a rapid spread of the infection once it was established. Based upon quantitative reverse transcription polymerase chain reaction data, we characterized the expression of 12 NBS-encoding genes in response to infection by A. alternata. We further characterized the structure, chromosomal location, and physico-chemical properties of the encoded proteins for these 12 NBS-encoding genes. Temporal gene expression profiles following inoculation indicated that the 12 NBS-encoding genes were all involved in the response in the two apple cultivars. The most highly expressed genes, MDP0000127009, MDP0000292810, and MDP0000710999, appear to have great potential for breeding resistant apple cultivars through genetic engineering.