Abstract: Valine-glutamine (VQ) is a plant-specific protein, which plays an important role in plant growth, development, and resistance to biotic and abiotic stresses. To date, however, little research has been conducted on the VQ gene family in perennial ryegrass (Lolium perenne L.). In this study, 52 LpVQ genes were identified in the perennial ryegrass genome, which were unevenly distributed on seven chromosomes. Subcellular localization prediction indicated that LpVQ proteins were mainly located in the nucleus, with only LpVQ45 located in the cell membrane. Phylogenetic analysis showed that LpVQ genes were divided into seven subfamilies and there were only four pairs of lineal homologous genes with Arabidopsis. Based on gene structure analysis of the LpVQ gene family, 88.46% of LpVQ genes had no intron. Finally, three LpVQ genes were randomly from each subfamily to observe the responses to dark, high temperature, and salt stress. The qRT-PCR analysis LpVQ genes responded quickly in early stages of stress. This study provides a theoretical foundation for further study on VQ family genes of perennial ryegrass under abiotic stress.