Parvovirus B19 (B19V), a single-stranded DNA virus in the family Parvoviridae, is a human pathogenic virus, characterized by a selective but not exclusive tropism for erythroid progenitor cells. Widely diffuse, it is responsible for an ample range of clinical manifestations, whose characteristics and outcomes depend on the interplay between the viral properties and the physiological and immune status of the infected individuals. The complexity of virus-host relationship and the diversity of the clinical course of infection pose a diagnostic challenge that may require non-trivial solutions. Areas covered: The review includes an updated description of the course of B19V infection in its complexity and diversity of pathogenetic mechanisms, discusses the consequent requirements for different and appropriated diagnostic approaches, presents the main diagnostic techniques, more recent technical advancements, and their application to the diverse clinical situations. Expert commentary: The complex scenario of the infectious process and the diversity in possible pathogenetic mechanisms make necessary a multi-parametric approach for an accurate and informative laboratory diagnosis of B19V infection, combining as much as possible the molecular detection of viral components, mainly viral DNA, to commonly followed immunological detection of virus-specific antibodies and a critical assessment of laboratory findings.