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Intramuscular fat (IMF) is vital for meat tenderness and juiciness. This study aims to explore the IMF deposition mechanism and the related molecular markers in sheep. Two populations, Small-tail Han Sheep (STH) and STH × Suffolk (SFK) F₁ (SFK × STH), were used as the research object. Histological staining techniques compared the differences in the longissimus dorsi muscle among populations. A combination of transcriptome sequencing and biological information analysis screened and identified IMF-related target genes. Further, sequencing technology was employed to detect SNP loci of target genes to evaluate their potential as genetic markers. Histological staining revealed that the muscle fiber gap in the SFK × STH F₁ was larger and the IMF content was higher. Transcriptome analysis revealed that <i>PIK3R1</i> and <i>PPARA</i> were candidate genes. Histological experiments revealed that the expressions of <i>PIK3R1</i> mRNA and <i>PPARA</i> mRNA were lower in SFK × STH F₁ compared with the STH. Meanwhile, PIK3R1 and PPARA proteins were located in intramuscular adipocytes and co-located with the lipid metabolism marker molecule (FASN). SNP locus analysis revealed a mutation site in exon 7 of the <i>PIK3R</i>1 gene, which served as a potential genetic marker for IMF deposition. This study’s findings will provide a new direction for meat quality breeding in sheep.