Morphological evaluation of cerebrospinal fluid (CSF) samples by light microscopy is the method of choice for the detection of central nervous system involvement in patients with haematological malignancies. In this retrospective study, we assessed the value of three-colour immunophenotyping by flow cytometry in addition to conventional cytology in 28 patients with and 17 patients without pre-existent haematological malignancy in whom CSF analysis was performed because of neurological abnormalities (n = 37) or as part of routine staging procedures (n = 8). Four samples could not be analysed by flow cytometry because of insufficient cell numbers. CSF involvement was detected in a total of 18 patients, in 12 by both cytology and flow cytometry, and in three each by either cytology or flow cytometry alone. Discordant results were particularly frequent in patients with low CSF cell numbers and B cell neoplasms. When combined with conventional cytology, flow cytometric analysis improves the detection rate of CSF involvement in patients with haematological malignancies. It is particularly useful in B cell neoplasms with low CSF cell numbers.