The prognostic value of DNA ploidy and proliferative index (PI) are well established in many cancers, but their significance in childhood rhabdomyosarcoma (RMS) is unclear. We studied the DNA content and PI of 45 cases of childhood RMS obtained retrospectively. DNA histograms were hyperdiploid in 30 cases (67%), diploid in 6 (13%), tetraploid in 5 (11%), polyploid in 3 (7%), and nonclassifiable in 1 (2%). The 5-year overall survival rate by ploidy was 60% (3/5) in tetraploid, 57% (17/30) in hyperdiploid, and 0% in diploid and polyploid cases (P = .000002). The 5-year overall survival by a PI less than or greater than 19% was 62% (13/21) and 21% (5/24), respectively (P = .006). In multivariate analysis, DNA ploidy (P = .001) was an important independent prognostic factor. DNA content in childhood RMS is an important variable in predicting prognosis. DNA hyperdiploid and tetraploid rhabdomyosarcomas had a favorable prognosis, while DNA diploid and polyploid tumors had a poor prognosis.