Infection of pigs with swinepox virus (SWPV) was reported in Brazil in 2011. SWPV causes a systemic pustular disease in pigs and the symptoms are clinically indistinguishable from those caused by vaccinia virus (VACV) infection. Pigs infected with VACV have been reported in various countries; however, VACV is endemic in Brazil, India and other countries, where it affects mainly dairy cows, dairy buffaloes and dairy workers causing localized pustules. The transmission of VACV to other susceptible hosts has also been detected in Brazil. Therefore, VACV should be investigated as a possible etiologic agent of pustular skin disorders in pigs. This work describes the development of a one-step duplex assay to detect swinepox and vaccinia viruses simultaneously in skin lesions of pigs with generalized pustular disease. The investigation of VACV infection in pigs is important in countries where this zoonosis is endemic and should be differentiated from SWPV infection.