Thirty one tracheal swabs were collected from diseased broiler chickens experiencing respiratory signs and congestion of head and shank regions and molecularly characterized in Sohag province. The results showed, twenty five (80.6%) of swabs were confirmed positive for carrying type A Avian influenza virus (AIV) by using rapid antigen detection kit. Total RNAs were extracted from the 17 positive AI type A swabs and One step RT-PCR test was performed for subtyping using specific primers targeting H5, H7, N1 and N2. AIV H5 subtype was detected in 9 samples and the rest was untypeable with H7 primers. From the 9 H5 strains, one H5N1 detected and 3 were H5N2 that reflects the presence of two subtypes co-circulated at that time of infection and other untypeable strains with our available primers. Molecular pathotyping was carried on the fully characterized H5 strains using Restriction Enzyme Cleavage Pattern (RECP) assay with the aid of MboII restriction enzyme as a new rapid method to avoid the disadvantages of reliable conventional methods (pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and sequencing of hemagglutinin (HA) gene cleavage site). The results revealed that 3 out of 4 (75%) examined H5N1 and H5N2 viruses were pathogenic and one H5N2 strain was low pathogenic. The SDS-page profiling revealed that the examined field isolates were distinct to two clusters; the first one having structural polypeptide: Haemagglutinin (61 KD) and (89KD) protein and the second cluster contains 61 KD, 89KD protein and 50 KDa neuramindase protein. In addition the western blotting showed that the killed Chinese H5N1 vaccine was used in Sohag province by farmers was of low antigenicity which the antibodies reacted only with haemagglutinin protein (61KDa) in all examined strains and one strain reacted with a high molecular weight protein (89KDa). Our data provide proof for the prevalence of H5N2 subtype than H5N1 as well as untypeable strains and low antigenicity of the used vaccine that reflects the low efficiency of the used vaccine in Sohag. So, the field strains should be updated in a timely manner through surveillance and accompanying laboratory evaluation of contemporary viruses for antigenic similarity with existing vaccine strains and so production of good immunity.