Due to its high rate of homologous recombination, the moss Physcomitrella patens (Hedw.) B.S.G. is used as a novel system to facilitate gene/function-analyses. Loss-of-function mutations are easy to identify in the moss, as the dominant phase is the haploid gametophyte. Regenerating protoplasts were used to establish different moss lines with ploidy levels of 1C, 2C, and 4C. Flowcytometric analysis of three haploid, three diploid, and two tetraploid Physcomitrella lines revealed that haploid and diploid lines were cytologically stable, whereas nuclei of tetraploid lines exhibited varying DNA-contents. The effect of polyploidization on the phenotype, growth, and differentiation of Physcomitrella was investigated in vitro. The growth of three haploid, three diploid, and two tetraploid genotypes was evaluated after four weeks of axenic culture. The effect of the genotype on the growth rate of plants cultured on minimal medium was statistically significant; in contrast the ploidy level had no effect. On full medium the effect of the line, as well as the ploidy level, were statistically significant. The dry weight per petri-dish after a culture period of four weeks, was 103–106 mg in haploid lines, 64–78 mg in diploid, and 19–32 mg in tetraploid ones. The effect of the moss line and the ploidy level on differentiation of buds and gametophores was highly significant. Tetraploid moss rarely developed buds and/or gametophores. Sporophyte induction was observed in haploid and diploid genotypes, however not in tetraploid moss. Twelve percent of the germinated spores increased the ploidy level in comparison with the parent plant. A reduction of the ploidy level from 2C to 1C was observed in 7%. Evaluation of older plants (11 weeks after protoplast isolation) showed that polyploidization often resulted in a reduced number of gametophores and a reduced colony diameter on minimal medium compared to haploid plants. More than 70% of the diploid plants had gametophores with phenotypic alterations. Only 1.4% of the diploid plants were indistinguishable from wildtype. Changes in leaf shape and multiple phenotypic deviations from wildtype are most likely indications of polyploidization; however, the unequivocal identification of diploid plants is only possible using flowcytometric analysis, because a multitude of different phenotypic changes were observed in polyploid plants.