Newcastle disease (ND) is a highly devastating disease for the poultry industry as it causes high economic losses. In this present study, a DNA vaccine containing the F and HN surface antigens of a highly virulent Newcastle disease virus (NDV), NDV/1/Chicken/2005 (FJ939313), was successfully generated. Cell transfection test indicated that the vaccine expressed the F and HN genes in Hep-2 cells. The main objective of this study was to compare the extent of protection induced by DNA vaccination after homologous and heterologous NDV-challenge as determined by the amount of NDV shedding after challenge. NDV-antibody-negative chickens were vaccinated either once, twice or thrice intramuscularly at 7, 14 and 21 days old and were challenged 14 days post vaccination with either homologous virus (vaccine matched velogenic viscerotropic Newcastle disease virus (vvNDV) strain, FJ939313), phylogenetically related to group VII, or a phylogenetically divergent heterologous virus (unmatched vvNDV strain, AY968809), which belongs to genogroup VI and shows 84.1% nucleotide similarity to the NDV-sequences of the DNA vaccine. Our data indicate that birds, which received a single dose of the DNA vaccine were poorly protected, and only 30–40% of these birds survived after challenge with high virus shedding titre. Multiple administration of the DNA vaccine induced high protection rates of 70–90% with reduced virus shedding compared to the non-vaccinated and challenged birds. Generally, homologous challenge led to reduced tracheal and cloacal shedding compared to the heterologous vvNDV strain. This study provides a promising approach for the control of ND in chickens using DNA vaccines, which are phylogenetically closely related to the circulating field strains.