By means of reverse phase and ion-exchange high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MAIDI-TOF MS) and amino acid sequence analysis of peptides, the venom from the spider found in Tongshi county, Hainan province of south China was analyzed and compared with that of the spider Selenocosmia huwena. Evident differences were found in the HPLC chromatograms and MAIDI-TOF spectrum of the venom from the two spiders. The amino acid sequences of the main neurotoxin from the two spiders are also different. The results indicated that, although the two spiders are similar to each other morphologically, they should belong to different species. The spider from Tongshi, Hainan was identified as a new species and was named Selenocosmia hainana sp. nov. Type specimens are deposited in College of Life Sciences, Hunan Normal University.