Five Liphistius Schiödte, 1849 species of the primitively segmented spider family Liphistiidae are currently known from Myanmar. Here, we described a new species, Liphistius pyinoolwin sp. nov. (♂♀), which was collected from Pyin Oo Lwin, Mandalay Region, Myanmar, diagnosed based on its genital morphology. The specimens (2♂♂, 5♀♀) collected by Walter C. Sedgwick from Pyin Oo Lwin in 1982 were misidentified as L. birmanicus Thorell, 1897 and are treated here as the newly described species. Accordingly, we described the males of L. birmanicus for the first time, redescribed its female, using newly collected specimens from Yadò, Than Taung and Kalekho Atet townships, Kayin State. We also provided information on the variation in genital morphology of both species, and their relationships within the birmanicus-group of species.