Genetic polymorphisms of CYP2C9 significantly influence the pharmacokinetics and pharmacodynamics of some drugs, which might result in adverse drug effects and therapeutic failure. Several studies have been performed on CYP2C9 genetic polymorphisms in Han Chinese populations. However, these studies only focused on two commonly investigated alleles, *2 and *3, in relatively small sample sizes. To scale up the gene-scanning region and determine relatively precise data on the genetic distribution pattern in Chinese populations, unrelated healthy Han Chinese volunteers from Zhejiang Province (n=1127) and Hebei (n=1000) Province were recruited as subjects for the direct sequencing of all exons of CYP2C9. As a result, 14 previously reported alleles were detected in this work, and 8 of these alleles (*14, *16, *19, *23, *27, *29, *33 and *34) were described for the first time in Chinese populations. In addition, 37 novel mutations were also detected, of which 22 variants were non-synonymous, and 21 new alleles, *36-*56, were designated by the Human CYP Allele Nomenclature Committee. In vitro functional analysis of these 22 novel CYP2C9 variants revealed that 17 mutations had a significant influence on the protein's catalytic activity. Our study provides the most accurate data on CYP2C9 polymorphisms in Han Chinese populations and detects the largest number of novel allelic variants existing to date. These new alleles will greatly enrich the current knowledge of naturally occurring CYP2C9 variants in Chinese populations.