Different serotypes of Haemophilus influenzae is now divided into 2 divisions: encapsulated and unencapsulated. Multiple locus variable number tandem repeat analysis (MLVA) includes such specifications as the extra power of separation, ease of data interpretation, and epidemiological data accordance, which have made it an appropriate molecular device for good typing and phylogenetic analysis of bacterial pathogens.In this research, cultured samples were studied and strains identified through biochemical tests were recognized. Moreover, DNA was extracted and studied qualitatively and quantitatively. Four pairs of specialized primers related to H. influenzae variable number tandem repeats (VNTR) and preparation of PCR were designed according to the regulated program. Also, electrophoresis of PCR products was performed. Finally, the interpretation of electrophoresis gel was done with respect to the observable bands showing the presence or absence of the required sequence in the samples related to every primer.This study was the first MLVA typing of the unencapsulated H. influenzae in Iran. In this research, the VNTR sequences were tested in 30 strains without the unencapsulated H. influenzae. Among 30 mentioned strains, for which MLVA profile was obtained in this research, 25 different MLVA types were observed. Likewise, there was no repetition in VNTR sequences resulting from PCR in few H. influenzae. In all these cases, the number of repetitions in MLVA profile was determined as 0, except for one of the primers in 4 strains, which was 16%. However, this did not occur for the other VNTRs.The highest diversity of the repeats was for VNTR5 (7 types), followed by VNTR6 with 6 types of repeats, and VNTR12-1 and VNTR12-2 with 3 different types.