Pigeon pox virus causes a serious disease in pigeons and may threats the life of these birds. The pigeon pox virus is easily detected in clinical samples when the signs appeared but in that case the disease could affect the health of birds and may be complicated by secondary bacterial infections. So, the rapid and accurate detection of the virus is needed. In this study the application of molecular techniques based on nucleic acid was characterization done. Molecular characterization of pigeon pox virus was successful carried out by using PCR and real-time PCR techniques for the pox virus in samples from 8 clinical cases of suspected diseased pigeons from three provinces (Giza, Kafr El-Sheikh and Beni-Suef). DNA was extracted from skin lesions from each case, the primers and PCR conditions were used to amplify the FWPV P4b gene. Results of PCR amplification of pigeon poxvirus and agarose gel electrophoresis showed that there were five out of the eight suspected field samples were positive for the presence of pigeon pox virus with the expected correct size bands of 578 bp. Real-time polymerase chain reactions (r-PCR) assay was also used for detection of the virus by using the same previously described primers with SYBR Green mix. Real time PCR dissociation curve of PCR products of the SYBR Green PCR assay indicated that the PCR products of melting temperature (Tm) at 75-77 °C were positive for 5 samples out of the 8 suspected cases of pigeon pox virus similar to the obtained results by conventional PCR. In this study, the molecular methods used were able to detect virus in clinical samples and showed the same sensitivity as virus isolation in case of obvious clinical signs but the molecular methods for virus isolation.