BACKGROUND: RNA sequencing (RNA-seq) is a powerful tool for transcriptome profiling, enabling integrative studies of expression quantitative trait loci (eQTL). As it identifies fewer genetic variants than DNA sequencing (DNA-seq), reference panel-based genotype imputation is often required to enhance its utility. RESULTS: This study evaluated the accuracy of genotype imputation using SNPs called from RNA-seq data (RNA-SNPs). SNP features from 6,567 RNA-seq samples across 28 pig tissues were used to mask whole genome sequencing (WGS) data, with the Pig Genomic Reference Panel (PGRP) serving as the reference panel. Three imputation software tools (i.e., Beagle, Minimac4, and Impute5) were employed to perform the imputation. The result showed that RNA-SNPs achieved higher imputation accuracy (CR: 0.895 ~ 0.933
r²: 0.745 ~ 0.817) than SNPs from GeneSeek Genomic Profiler Porcine SNP50 BeadChip (Chip-SNPs) (CR: 0.873 ~ 0.909
r²: 0.629 ~ 0.698), and lower accuracy in "intergenic" regions. After imputation, quality control (QC) by minor allele frequency (MAF) and imputation quality (DR²) could improve r² but reduce SNP retention. Among software, Minimac4 takes the least runtime in single-thread setting, while Beagle performed best in multi-thread setting and phasing. Impute5 takes up minimal memory usage but requires the maximum runtime. All tools demonstrated comparable global accuracy (CR: 0.906 ~ 0.917
r²: 0.780 ~ 0.787). CONCLUSIONS: This study offers practical guidance for conducting RNA-SNP imputation strategies in genome and transcriptome research.