Tissue culture‐induced variants (somaclones) of reed canarygrass (Phalaris arundinacea L.) were developed from callus cells of aseptic spikelet cultures. Molecular polymorphism of the populations of somaclones and spikelet‐donor plants as natural vegetative clones were compared by the application of polymerase chain reaction (PCR)‐based techniques of random amplified polymorphic DNA (RAPD)‐, simple sequence repeat (SSR)‐, and inter‐simple sequence repeat (ISSR)‐PCR to genomic DNA samples. The genetic diversity was higher in the somaclones compared with vegetative clones as measured by the maximum genetic distance (MaxGD) which increased from 0·401 (vegetative clones) to 0·498 (somaclones). Along with this change, the minimum genetic distance (MinGD) decreased from 0·20 (control plants) to 0·06 (somaclones) which demonstrated that genetic changes occurred not only in a positive (dissimilarity, MaxGD), but also in a negative (similarity, MinGD) direction. Structural carbohydrate analyses were also performed on the leaves to compare the somaclones with the donor vegetative clones. The mean neutral‐detergent and acid‐detergent fibre concentrations of the leaves of the somaclones were about 0·20 lower than the donor vegetative clones suggesting that increases in the digestibility could be obtained from the somaclonal material.