Protein disulfide isomerase (PDI) is an important enzyme for protein folding in endoplasmic reticulum. The PDI gene family has been systematically studied in Arabidopsis, barley, rice, and bread wheat; however, little is known about this gene family and their roles during fruit development and ripening in fruit trees. In this study, 63 PDI genes were isolated from 8 fruit trees. Phylogenetic trees showed that these genes were clustered into six different groups, designated as A to F. In the groups, the PDI genes had significant differences in gene structure and conserved regions. The chromosome location of each PDI gene was determined in complete genome-assembly fruit trees and the synteny of chromosome segments was detected among peach, pear, and strawberry. Expression profiles of PDI genes in peach, pear, and strawberry showed that nearly all genes in group D and E were more highly expressed in developmental and ripening fruit tissues than those in other groups, while all genes in group A and B presented the lowest levels of expression in fruits of each stage. Moreover, qRT-PCR analyses revealed that these expressed genes were stable expressed in pear and peach fruits, as well as the reported reference genes. Eventually, PbPDI.F1 presented the highest expression stability in pear fruit while PpPDI.F displayed stronger stability than other genes in peach fruit. Thus, these two genes, which were clustered in group F, are good reference genes for gene expression studies during fruit development and ripening.