This study was focused on identifying the effects of single nucleotide polymorphisms (SNPs) located on an entire region of the let-7c miRNA gene with consideration of its ability to promote litter size in Markhoz goats. The Markhoz goat, the native breed in Iran, is important for its reproductive traits, such as litter size. DNA polymorphism of let-7c miRNA gene was revealed and considered for further studies for its effect on litter size in Markhoz goats. PCR-SSCP analysis investigated different band patterns for this miRNA; however, sequencing results have detected only an A to T substitution located five nucleotides downstream of the let-7c miRNA gene. The chi-squared test showed that the let-7c miRNA gene locus was out of the Hardy–Weinberg equilibrium (HWE) and has significant effect (p < 0.05). Furthermore, the least-square analysis indicated that the let-7c miRNA gene does not affect prolificacy in the Markhoz goat (p > 0.05). In sum, all loci failed to have a significant effect on the litter size trait (p > 0.05). Moreover, years of kidding and parity had no significant impact on let-7c_S (p > 0.05); however, the let-7c_B affected the litter size trait significantly (p < 0.05). Additionally, binary logistic regression and chi-square analysis revealed that allele A of the detected SNP within 3′ UTR region of the let-7c gene had a non-significant effect on litter size in the studied goats (p > 0.05).