Flavor is an essential characteristic of fruit quality and is significant for consumers. Off-flavors have been reported in several fruits, including sweet cherry. This fruit has been reported to show an herbaceous/grassy-like flavor. The herbaceous off-flavor in sweet cherries detected in cultivar Regina has been related to the differential development of aroma compounds such as short-chain aldehydes and esters. One of the main biosynthesis pathways for these compounds is the fatty acid oxidation mediated by lipoxygenases (LOX). In order to have a better understanding of the biological basis of the differences in the volatile profile, the LOX gene expression profile was characterized during fruit development with and without herbaceous off-flavor. A genome-wide analysis of LOX in sweet cherry was carried out and compared to other species such as Arabidopsis, tomato, apple, prunus and strawberry. The structural features of 9-LOX and 13-LOX genes, encoded protein domains and their synteny were examined. Moreover, we analyzed the LOX expression at four developmental stages along ripening by RT-qPCR. Thirteen LOX gene candidates (six 9-LOX and seven 13-LOX) were identified. The 13-LOXs, PaLOX10, PaLOX11, and PaLOX12 were differentially expressed in herbaceous sweet cherries. Furthermore, their expression profile positively correlated with key volatile compounds linked to the herbaceous off-flavor. Overall, this study involves the genome-wide characterization of the LOX family in Prunus avium cv. Regina and provides information that can aid in studying LOX-related fruit deterioration in sweet cherries and associated species.