Microspore-derived callus cultures were obtained by anther culture of `Emperor Francis' sweet cherry (Prunus avium L.). Branches were removed from the field in January and March and forced in the laboratory. When the microspores reached the uninucleate stage, anthers were placed on modified Quoirin and Lepoivre liquid culture medium containing 4.4 μm BA and 4.5 μm 2,4-D. After ≈60 days, callus that emerged from the anthers was placed on woody plant medium supplemented with 1 μm 2,4-D and 3 μm 2iP and routinely transferred. The resulting 270 callus cultures were screened for two allozymes heterozygous in `Emperor Francis', Pgi-2 and 6-Pgd-1. Of the 270 callus cultures, 154 expressed only one allele each for Pgi-2 and 6-Pgd-1; thus, they were considered microspore-derived. The microspore-derived callus cultures can be used as a linkage mapping population. Chemical names used: 6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); N6-(2-isopentenyl)-adenine (2iP).