Background: There is scarce information on Varicella–Zoster virus genetic variability in individuals with acute central nervous system infection in Brazil. The objective of this study was the molecular characterization of Varicella–Zoster virus isolates in cerebrospinal fluid from individuals with acute central nervous system infection. Methods: Cerebrospinal fluid samples were collected from individuals evaluated in emergency and community healthcare services in São Paulo, Brazil. Varicella–Zoster virus identification was performed using commercial platforms Biofire-FilmArray Meningitis/Encephalitis (BioMérieux, Craponne, France) and XGEN-UMLTI-N9^® (Mobius Life, Pinhais, Brazil). Positive samples were further characterized as wild-type or vaccine-strain by a real-time polymerase chain reaction assay that targeted a single nucleotide polymorphism in open reading frame 62. We also estimated the mean genetic distance and phylogenetic reconstruction based on open reading frames 22, 38, 54, and 62 in relation to sequences of intercontinentally circulating Varicella–Zoster virus isolates. Results: Among the 600 cerebrospinal fluid samples, we identified Varicella–Zoster virus in 30 (5%) samples. None were positive for the vaccine-strain. Twelve samples were sequenced and phylogenetically classified into Clades 1 (41.7%), 2 (25%), 3 (8.3%), 5 (16.7%), or 6 (8%). Conclusion: Enhanced characterization of circulating Varicella–Zoster virus Clades in Brazil identified previously unreported Clades 2 and 6 as well as three other Clades disseminated intercontinentally. These findings reinforce the importance of Varicella–Zoster virus molecular surveillance in cerebrospinal fluid.