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Single nucleotide polymorphisms (SNPs) are the most common genetic variations that can be genotyped effectively by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Although PCR-RFLP has become the popular technique, search for available restriction enzymes and design of feasible primers for SNP genotyping is still a challenging task. An available restriction enzyme at least must be provided to discriminate a target SNP, and simultaneously a feasible primer pair observes numerous constraints must be given before performing SNP-based PCR-RFLP experiments. Here, we called it "natural SNP-RFLP primer design". In the past, a memetic algorithm (MA) was introduced to design natural SNP-RFLP primers, however, the influence of the used population size was not considered. Here, we use different population size to estimate the result of MA-based natural SNP-RFLP primer design. From the test result, we suggested the population size used between 200 and 300 is preferred to provide the natural SNP-RFLP primers.