The most serious type of coronary artery disease (CAD), acute myocardial infarction (AMI), is a major global cause of death. The development of AMI is accompanied by several risk factors. AMI may be caused by variations in the microRNA (miRNA) genes, which have a negative impact on miRNA-mediated regulation of gene expression. The target mRNAs are dysregulated because of these genetic changes in the miRNA genes, which interfere with the vital biological processes that result in AMI. Using allele-specific PCR, the aim of the study is to examine the association of the variants (rs2910164, rs4636297, and rs895819) in MIR146A, MIR126, and MIR27A with AMI susceptibility. A difference in genotype distribution among the patients and control for variation rs2910164 was identified by co-dominant [χ2 = 68.34,2; P value<0.0001], dominant (G/G vs G/C + C/C) [OR = 4.167 (2.860–6.049); P value<0.0001], recessive (C/C vs G/C + G/G) [OR = 0.2584 (0.1798–0.3731); P value<0.0001], and additive models [OR = 3.847 (2.985–4.959); P value<0.0001]. Whereas the association of rs4636297 was investigated by co-dominant [χ2 = 6.882,2; P value = 0.0320], dominant (G/G vs G/A + A/A) [OR = 0.6914 (0.4849–0.9948); P value = 0.0489], recessive (A/A vs A/G + G/G) [OR = 2.434 (0.9849–5.616830); P value = 0.0595], and additive models [OR = 0.7716 (0.6000–0.9918); P value = 0.0433]. Similarly, association of rs895819 was determined by co-dominant [χ2 = 5.277, 2; P value = 0.0715], dominant (G/G vs G/A + A/A) [OR = 1.654(0.9819–2.801); P value = 0.06440], recessive (A/A vs A/G + G/G) [OR = 0.7227 (0.5132–1.022); P value = 0.0748], and additive models [OR = 1.3337 (1.041–1.719); P value = 0.0233]. The results of this study found a significant association of rs2910164 and rs4636297 with AMI and are considered as the risk factor for AMI in the Pakistani population. We observed no significant association of the variant MIR27A (rs895819) with AMI incidence.