MHinNGS is a Python application developed for analysis of microhaplotypes (MHs) in single-end sequencing data. MHinNGS analyses reads in standard formats and store each sequence into bins, one bin for each MH as defined by the two flanking sequences. MHinNGS requires a reference genome and a configuration file with information about each locus. Four mandatory and 15 optional criteria defined in the configuration file allow detailed locus-specific analyses of the MH loci. The program 1) removes noise, 2) identify and name alleles, 3) test the genotypes, and 4) test unique sequences not identified as noise or alleles. MHinNGS produces a result file, where every unique sequence that passed the noise filter is presented with MH allele, read depth, warning flags based on the genotyping criteria, sequence, heterozygote balance, and MH name. Furthermore, variation in other parts of the fragment that is not defined as SNPs in the MH, linked variants, or rare SNPs are listed in a separate column of the result file.