The identification of genes specifically expressed at a developmental stage would provide insights into many characteristic features of each of the developmental stages and introduction of molecular markers linked to important agronomic traits. However, the current methods employed to identify genes with specific expression are labor-intensive and suffer from high rates of false positives. Here, we employed a new and accurate reverse transcription-polymerase chain reaction method using annealing control primers (ACP) to identify genes that are preferentially expressed in juvenile or mature leaves of pears (Pyrus pyrifolia L.). We identified and sequenced four of these differentially expressed genes (DEGs). Basic Local Alignment Search Tool searches revealed that one was a hypothetical gene and three genes encoded protein with high similarity to the known genes. These DEGs were further characterized using real-time quantitative PCR to assess their expression pattern. This report suggests that the ACP system is a rapid and accurate method for the identification of development-specific genes in woody plants such as pears, of which a few genes are available.