Abstract: Using GC-MS and IRMS testing technologies, the paper analyzes oil seepages and bitumen, correlates their maturity and oil sources,and discusses hydrocarbon accumulation history in Lower Ordovician Honghuayuan formation in southern Guizhou depression. The result shows that the oxidized bitumen in Honghuayuan formation is mainly found in Danzhai-Majiang area in the southern study area; while the Kaili area in the northern study area with oil shows occuing widely, oil shows occur widely, and is of mixture source characteristics by the fact that the major source rocks appear in Niutitang formation of the Lower Cambrian, the secondary hydrocarbon source rocks are found in Wengxiang shale of the Middel-Lower Silurian. Under the influence of multi-stage tectonic activities, the hydrocarbon accumulation history is relatively complex and featured with accumulation-destruction of multicycles, among which the destruction is the strongest during the late Yanshan Movement period