Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury. The acute alcoholic liver injury models were established by the intragastric administration of alcohol (5 mg/kg) in Imprinting Control Region mice. Total RNA was isolated from the mouse livers, following which the expression levels of seven reference genes, β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gadph), glucuronidase β, hypoxanthine phosphoribosyltransferase 1 (Hprt1), 18S ribosomal RNA, TATA binding protein and β‑2 microglobulin, were examined, and gene expression stability was assessed using the geNorm, NormFinder and BestKeeper tools. The geNorm analysis revealed that the gene with the lowest variability was Hprt1. Hprt1 and Gapdh were validated as the optimal reference gene pair in all samples from all groups. The NormFinder and BestKeeper results showed that Hprt1 was the most stable gene in all samples. Alcohol induces endoplasmic reticulum (ER) stress, causing changes in the expression levels of ER stress‑associated genes. The stability of Hprt1 was verified by the expression analysis of ER stress‑associated genes, and gene expression levels in the ethanol groups were upregulated, with a significant difference in expression, compared with those in the control group. Therefore, Hprt1 was selected as the most stable gene, and Hprt1 and Gapdh were determined to be the optimum gene pair in mouse models of acute alcoholic liver injury. The reliability of the Hprt1 gene was confirmed by expression analysis of ER stress‑associated genes.