Pathogenic variants in DCX on the X chromosome lead to lissencephaly and subcortical band heterotopia (SBH), brain malformations caused by neuronal migration defects. Doublecortin (DCX) binds to microtubules to modulate microtubule polymerization. How pathogenic DCX variants affect these activities remains not fully investigated. Our aim is to examine whether these variants affect microtubule binding and dynamics and develop a verification tool to apply on clinical research. DCX variants were identified using whole exome and Sanger sequencing from six families with lissencephaly/SBH. We examined how these variants affect DCX functions using microtubule binding, regrowth, and colocalization assays. We found DCX variants p.Val177AlafsTer31, p.Gly188Trp, p.Arg196His, p.Lys202Met, p.Thr203Ala and p.Asp262Gly. All of the variants are located on its C-terminal microtubule binding domain. The microtubule binding ability was significantly decreased in these variants. Furthermore, expression of DCX variants affected microtubule growth in cells. The microtubule colocalization in some of DCX variants also decreased. By in utero electroporation, we first overexpressed one DCX variants in mouse embryo, and found it affected neuronal migration. Our results indicate that these variants in the C-terminal DCX domain defected microtubule binding and dynamics, which may underlie neuronal migration defects during brain development.