A multiplex nested polymerase chain reaction (PCR) was developed for the detection of and differentiation between porcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen samples were found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the 30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight for PCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzed using multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cell fractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay was found to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection of and differentiation between PCV-1 and PCV-2 in boar semen.