Two Solanum brevidens specific repetitive DNA clones (pSB1 and pSB7) were used simultaneously as probes in fluorescence in situ hybridization (FISH) for cytological studies of somatohaploids and their somatic hybrid donors. pSB1 was labelled with digoxigenin-11-dUTP and pSB7 was labelled with biotin-14-dATP and they were detected with reporter molecules conjugated to fluorescent dyes using digital imaging. The tandemly repeated sequences hybridized mostly near the telomeres of the chromosomes of S. brevidens. Using these two probes, it was possible to identify chromosomes containing repetitive DNA of S. brevidens both in the somatic hybrids between S. brevidens and S. tuberosum, and somatohaploids derived from the somatic hybrids. These cytological analyses showed that for the largest part genomes of the hexaploid somatic hybrids and their anther-derived triploid somatohaploids were composed of the genome of S. brevidens.