Primer-introduced restriction analysis is widely used in molecular genetics. However, several studies have reported inconsistent data regarding sequencing, mainly among heterozygous samples. This discrepancy may be related to the bias towards a 1:1 ratio typically observed in heterozygous digestion products. In this study, we investigated the mechanism and minimization of this observed bias. Three mismatched polymerase chain reaction (PCR) models were analyzed by testing different PCR conditions and reaction mixtures. For EPHX1 gene rs1051740 single-nucleotide polymorphism PCR, DNA concentration, denaturation and elongation time, annealing temperature, and cycle number significantly influenced product ratios. For SERPINA1 gene PIMmalton deletion (ΔPhe52) and CHRNA3 gene rs1051730 single-nucleotide polymorphism PCRs, significant bias fluctuations were observed only for the annealing temperature and cycle number conditions. The relevance of these results to the amplification efficiency parameter is discussed. Rather than reducing the observed bias, our data provide evidence of a counterbalance for preferential amplification, depending on cycle number, annealing temperature, and amplification efficiency alteration. Our results are relevant for application to primer-introduced restriction analysis PCR assays.