Traditionally,strain types of bacteria are determined by multilocus sequence typing(MLST)of several housekeeping genes.In case of mixed population of nonculturable bacteria,generation of larg.e clone libraries and the Sanger sequencing of each clones are additionally required.In the next-generation sequencing(NGS)era,most of the sequencing experiments have been replaced by the NGS techniques.Thus,in line with the new sequencing trend,the feasibility of pyrosequencing-based MLST was assessed in this study.We tried typing of one of the famous endosymbiont,namely Wolbachia.Wolbachia is an obligate symbiotic bacteria which is nonculturable and found in 70%of insect species.We collected three species of butterflies in Korea(Eurema hecabe,Eurema laeta,and Tongeia fischeri)as test organisms.Six genes(ftsZ,gatB,coxA,hcpA,fopA,and wsp)were investigated for comparing Wolbachia strain types by Sanger and pyrosequencing.The results demonstrate that there were several limitations with the pyrosequencing-based MLST.Firstly,the short sequence length of pyrosequencing reads hindered the complete identification of the long(>500 nt)sequence of wsp.Secondly,the time and cost effect of NGS-MLST is inferior to Sanger-based MLST.Thirdly,strain typing may not possible if the sequence type is a new one that has not been registered in public database,though that case was not observed in this study.However pyrosequencing-based MLST has a higher sensitivity than cloning and Sanger sequencing methods for the detection of minor alleles.Considering the high prevalence of infection with multiple Wolbachia STs,next-generation sequencing with improved analysis would assist with scaling up approaches to Wolbachia MLST.