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Reverse transcription and real time PCR method (rRT-PCR), according to Wise et al. (2004), and virus isolation were used for detection of Newcastle disease virus (NDV) in domestic poultry and wild birds in Bulgaria during 2005 and 2006. Six isolates from chickens, turkeys and a swan were investigated in this study. Nucleotide sequence analysis and deduced amino acid sequence at the cleavage site of the F protein showed that all of the isolates derived from domestic poultry were velogenic and they all had the cleavage site motif of RRQKRF. In contrast, the isolate from the swan was lentogenic NDV with GRQGRL motif. The velogenic isolates belong to the genetic lineage 5d, whereas the lentogenic isolate belongs to the genetic lineage 2. The rRT-PCR method detected viral RNA from both cloacal and tracheal swabs, and also from corresponding isolates, i.e. allantoic fluids of inoculated chicken embryos. The results from the tracheal swabs always showed earlier Ct value and higher fluorescence compared to the cloacal swabs. Therefore, tracheal swabs should be sampled and tested for detection of ND in poultry and wild birds. Although the F gene primers and probe were originally created to specifically detect mesogenic and velogenic NDVs’ from California 2002-2003 outbreak, they could detect all velogenic isolates from Bulgaria during 2005 and 2006.