Abstract Purpose Neuroblastoma (NB) is the most frequent extracranial tumor in children. The detection of bone marrow (BM) involvement is crucial for correct staging and risk-adapted treatment. We compared three methods regarding the detection of NB involvement in BM. Methods Eighty-one patients with NB were included in this retrospective study. BM samples were obtained at designated time points at study entry and during treatment or follow-up. The diagnostic tools for BM analysis included cytomorphology (CM), flow cytometry (FCM) and automatic immunofluorescence plus fluorescence in situ hybridization (AIPF). Results We analyzed 369 aspirates in 81 patients in whom AIPF, CM, and FCM were simultaneously available. During the observation period, NB cells were detected in 86/369 (23.3%) cases, by CM in 32/369 (8.7%), by FCM in 52 (14.1%), and by AIPF in 72 (19.5%) samples. AIPF and/or FCM confirmed all positive results obtained in CM and detected 11 additional positive BM aspirates in 294 CM negative samples (p < 0,001). Survival of patients with BM involvement at study entry identified solely by FCM/AIPF was 17.4% versus 0% for patients in whom BM involvement was already identified by CM. Conclusion The combination of AIPF/FCM yielded the highest detection rate of NB cells in BM. AIPF was the single, most sensitive method in detecting these cells. Although CM did not provide any additional positive results, it is still a useful, readily available and cost-effective tool. The prognostic significance of FCM and AIPF should be confirmed in a prospective study with a larger number of patients.