Humamrodent somatic cell hybrids carrying a single, intact, selectable human chromosome are valuable both for functional somatic cell genetic analysis and genome mapping procedures. Here, we describe the construction and detailed molecular cytogenetic characterization of a panel of 23 stable hybrids, representing all 22 human autosomes plus the X-chromosome. Individual normal human chromosomes have been tagged with a selectable fusion gene (Hytk) introduced into the chromosome in a small (4.2 kbp) retro viral vector. Use of the Hytk marker permits both positive and negative (“in-out”) selection to be applied to the human chromosome in any mammalian cell background. The panel includes 18 new hybrids isolated by direct microcell transfer from normal human diploid fibroblasts into mouse A9 cells.