Tarsal scopula condition and carapace length were studied for eighteen Ischnocolinae species. For cladistic analysis a matrix of 20 terminals and 30 characters of representatives of Ischnocolinae, Theraphosinae, Aviculariinae, Harpactirinae and Trichopelmatinae were analyzed using Nona 2.0 computer software. The matrix was analyzed in four different ways: 1. each tarsal scopula (legs I–IV) coded as separate characters; 2. one character with six ordered states; 3. one character with six independent states; 4. without tarsal scopula character. The first two matrices result in one tree with the same indices (L = 72; CI = 0.54; RI = 0.74) and topology: Part of Ischnocolinae is monophyletic (H. rondoni(S. longibulbi(I. algericus Catumiri))) and the other representatives (Oligoxystre and Genus 1) form a distinct monophyletic group with Theraphosinae, Harpactirinae and Aviculariinae. There are no homoplasies in tarsal scopula evolution in the second cladogram. The other two cladograms show less resolution for the Ischnocolinae than the two first cladorams. The tarsal scopula condition appears to have no relation to spider size (t =−0.80433; P = 0.438247) and should be used in phylogenetic analysis of Ischnocolinae because it provides information on the character variability within the subfamily.