Newcastle disease is economically the most important disease of poultry. The causative agent Newcastle disease virus (NDV) is a large, enveloped virus containing single stranded non-segmented negative-sense RNA genome. The genome of NDV contains six genes in the order of 3'Leader-N-P-M-F-HN-L-5'Trailer. NDV has at least three different genome size categories: 15,186, 15,192 and 15,198 nucleotides (nt) in length. The virulence of NDV is considered to be contributed by multiple genes. The importance of genome lengths and the roles of individual genes in virulence of NDV in its natural host, chickens, have not been determined. In this study, the effects of naturally occurring nucleotide insertions in NDV genome and roles of individual genes in the virulence of NDV in chickens were determined. To achieve this goal, reverse genetic systems for two strains of NDV were established for a highly virulent strain Texas GB (GBT) and a moderately virulent strain Beaudette C (BC). Both GBT and BC are isolated from chickens and belong to genotype II of class II NDV strains and have the genome length of 15,186 nt. The 6- and 12-nt insertions in the backbones of rBC and rGBT showed little attenuation in virus replication and in pathogenicity of the parental recombinant viruses. The reciprocal swap between NDV strains BC and GBT for the genes, nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large polymerase (L) protein genes, showed that F protein gene is most important for NDV virulence, followed by the L protein gene. M, HN, N and P genes appeared not to affect the pathotypes of their parental recombinant viruses in chickens. The observations of the present study paves the way for future directions: to use the naturally occurring insertion site in the coding region of the phosphoprotein gene for insertion of potential marker sequences; to determine the amino acid residues important in fusion protein and polymerase protein for replication and pathogenesis of NDV.