Complementation analysis of the combined Condit/Dales collection of vaccinia virus temperature-sensitive mutants has been reported (Lackner, C.A., D'Costa, S.M., Buck, C., Condit, R.C., 2003. Complementation analysis of the Dales collection of vaccinia virus temperature-sensitive mutants. Virology 305, 240-259), however not all complementation groups have previously been assigned to single genes on the viral genome. We have used marker rescue to map at least one representative of each complementation group to a unique viral gene. The final combined collection contains 124 temperature-sensitive mutants affecting 38 viral genes, plus five double mutants.