Abstract: Paternity testing is a technique of great importance in the genetic breeding of aquatic animals. Currently, the most frequently used type of biomarker in paternity tests is microsatellites (SSRs). However, weaknesses of SSRs lie in the complicated and labor-intensive genotyping process, which leads to low efficiency when such analyses are performed on a large scale. In this study, a new type of molecular biomarker, microhaplotypes (MH), was introduced for paternity testing. For the purpose of marker screening and paternity testing, a more efficient pipeline was constructed and evaluated with data from a grass carp population. The results showed that the genotypes of the MHs can be accurately obtained from genome resequencing data with clearly improved efficiency and compatibility over conventional genotyping methods based on population genetics. It is feasible to screen highly efficient MH combinations using the informative index. The consistency with the paternity test results obtained using SSRs reached 97.08% or 99.42% when 3 or 5 MHs were used, respectively. This research suggests that MHs can be used for the rapid and accurate paternity testing of fishes.