The ABA/water stress/ripening-induced (ASR) gene family exists universally in higher plants, and many ASR genes are up-regulated during periods of environmental stress and fruit ripening. Although a considerable amount of research has been performed investigating ASR gene response to abiotic stresses, relatively little is known about their roles in response to biotic stresses. In this report, we identified five ASR genes in apple (Malus × domestica) and explored their phylogenetic relationship, duplication events, and selective pressure. Five apple ASR genes (Md-ASR) were divided into two clades based on phylogenetic analysis. Species-specific duplication was detected in M. domestica ASR genes. Leaves of 'Golden delicious' and 'Starking' were infected with Alternaria alternata f. sp. mali, which causes apple blotch disease, and examined for the expression of the ASR genes in lesion areas during the first 72 h after inoculation. Md-ASR genes showed different expression patterns at different sampling times in 'Golden delicious' and 'Starking'. The activities of stress-related enzymes, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia lyase (PAL), and polyphenoloxidase (PPO), and the content of malondialdehyde (MDA) were also measured in different stages of disease development in two cultivars. The ASR gene expression patterns and theses physiological indexes for disease resistance suggested that Md-ASR genes are involved in biotic stress responses in apple.