Patients with parkinsonian symptoms can present either to primary care physicians or to neurologists. In both contexts, differential diagnosis is problematic, particularly early in the disease when only subtle bradykinesia, rigidity or tremor is present. Adjunctive tests should help substantially to improve the accuracy of early clinical diagnosis. This Review appraises cerebrospinal fluid (CSF), plasma and urine biomarkers that have been studied in the differential diagnosis of neurodegenerative parkinsonism. CSF biomarkers seem to hold the most promise because of their intimacy with the degenerating neurons. Most assays are still in the early stages of development, but CSF measures of alpha-synuclein (specific for Parkinson disease) and tau fragments (specific for progressive supranuclear palsy) have been refined. Universal approval of these assays will depend on larger clinical trials and establishment of normal ranges. Other blood and CSF biomarkers have shown exceptional specificity and sensitivity when analyzed in combination, although these findings require verification. A host of potential biomarkers have, however, produced disappointing results, either because of poor specificity or low assay reproducibility. Despite such difficulties, improved technology, in conjunction with advances in nosology and pathology, means that biomarkers are poised to enter routine clinical practice to aid the differentiation of parkinsonian disorders.