Identification of genetic variations is a central part of population and quantitative genomics studies based on high-throughput sequencing data. Even though popular variant callers such as Bcftools mpileup and GATK HaplotypeCaller were developed nearly 10 years ago, their performance is still largely unknown for non-human species. Here, we showed by benchmark analyses with a simulated insect population that Bcftools mpileup performs better than GATK HaplotypeCaller in terms of recovery rate and accuracy regardless of mapping software. The vast majority of false positives were observed from repeats, especially for GATK HaplotypeCaller. Variant scores calculated by GATK did not clearly distinguish true positives from false positives in the vast majority of cases, implying that hard-filtering with GATK could be challenging. These results suggest that Bcftools mpileup may be the first choice for non-human studies and that variants within repeats might have to be excluded for downstream analyses.