The 235kDa rhoptry protein (Py235) of Plasmodium yoelii is coded for by a multigene family. Py235 has been implicated in host cell selection and virulence as antibodies against it have been shown to inhibit invasion of mature red blood cells of the normally virulent P. yoelii YM line and at least one member of this family directly binds to erythrocytes. Differences in py235 sequence and copy number have been postulated to be responsible for the differences in invasion phenotype seen in the avirulent P. yoelii YA line and the YM line. The newly available sequence data for P. yoelii 17X NL 1.1 has now made it possible to investigate this further. A number of approaches including real time PCR was used to determine the exact copy number of individual py235. Except for two cases in YA and one in YM there are no differences in py235 copy number between the two lines and 17X NL 1.1. Analysis of progeny of a genetic cross between YM and an avirulent strain AC yield similar limited variations in copy number. This study shows that the copy number of py235 in the analyzed P. yoelii strains is significantly lower than previous estimates and much more in line with the published genome sequence. The lower copy number as well as the limited difference of py235 in the virulent lines makes it highly unlikely that these are the factors contributing to the differences in invasion observed.