In utero electroporation (IUE) is a useful technique for gene delivery in embryonic mouse brain. IUE technique is used to investigate the mammalian brain development in vivo. However, according to recent studies, IUE methodology has some limitations like the formation of artificial ectopias and heterotopias at the micro-injection site. Thus far, the artificial heterotopias generated by physical trauma during IUE are rarely reported. Here, we reported the artificial heterotopias and ectopias generated from surgical damages of micropipette in detail, and moreover, we described the protocol to avoid these phenotypes. For the experimental purpose, we transferred empty plasmids (pCAGIG-GFP) with green fluorescent-labelled protein into the cortical cortex by IUE and then compared the structure of the cortex region between the injected and un-injected cerebral hemispheres. The coronary section showed that ectopias and heterotopias were appeared on imperfect-injected brains, and layer maker staining, which including Ctip2 and TBR1 and laminin, can differentiate the physical damage, revealing the neurons in artificial ectopic and heterotopic area were not properly arranged. Moreover, premature differentiation of neurons in ectopias and heterotopias were observed. To avoid heterotopias and ectopias, we carefully manipulated the method of IUE application. Thus, this study might be helpful for the in utero electroporator to distinguish the artificial ectopias and heterotopias that caused by the physical injury by microneedle and the ways to avoid those undesirable circumstances.