Distribution microbiota of tropical-farmed Pangasius hypophthalmus fish during processing was examined by culture-dependent techniques and 16S rRNA gene sequencing. Two Vietnamese companies (BC: large scale factory, chlorine-based process, BW: large scale factory, water-based process and SC: small scale factory, chlorine-based process) processed Pangasius for intend-exporting to Western market was sampled. A total of 252 isolates originating from beginning, intermediate and final steps of the production line were then identified by 16S rRNA gene sequencing. The results of microbial profile showed no significant difference (p > 0.05) between two processing lines of BC and BW.  Surprisingly, chlorine treated fillets from the SC line were revealed to have significantly higher microbial counts than potable water treated fillets at BW line due to probably temperature abuse during processing. Along the processing chains, 131 of Gram negative and 43 of Gram positive bacteria were identified consisting of 20 different genera and 38 different species. Contrary to microbial counts, the type of spoilage related microbiota present on the BC and BW line was more diverse than that on the SC line.  Enterobacteriaceae (as spoilage and hygiene indicators) such as Providencia, Shigella, Klebsiella, Enterobacter and Wautersiella were isolated from fillets sampled on the SC line whereas Serratia was only observed on fillets sampled on the BC and BW lines. The results can be used to improve Good Manufacturing Practices for processed Pangasius fillets.