The human Y-chromosome haplogroup O-M134 is one of the most abundant paternal lineages in East Asian populations, comprising~13% of Han Chinese males, and also common in Kazakh, Korean, Japanese, Thai and so on. Despite its considerable prevalence, its current substructure is poorly resolved with only one downstream marker (M117) previously investigated. Here we address this deficiency by investigating some single-nucleotide polymorphisms (SNPs) previously reported being potentially associated with O-M134 based on high-throughput DNA-sequencing data. Using a panel of 1301 Chinese males we first identified 154 haplogroup O-M134 subjects. We then investigated the phylogenetic structure within this haplogroup using 10 SNPs (F444, F629, F3451, F46, F48, F209, F2887, F3386, F1739 and F152). Two major branches were identified, O-M117 and O-F444 and the latter was further divided into two main subclades, O-F629 and O-F3451, accounting for 10.84 and 0.92% of the Han Chinese, respectively. This update of O-M134 diversification permits better resolution of male lineages in population studies of East Asia.