The main objective of this study was to examine the existence of Relative Age Effect (RAE) and analyze the effect of gender on Fundamental Motor Skills (FMS) of preschool children aged 4-5 years old. A total of 298 healthy preschool children were evaluated. The Test of Gross Motor Development-2 (TGMD-2) was used to evaluate preschoolers' FMS. The data showed the main effects on the semester of birth factor in locomotor skills (LM) and total test score (TTS) (p < 0.05), but not in object control skills (OC; p > 0.05). There were also significant main effects on the gender factor in LM and TTS (p < 0.05), but not in OC (p > 0.05). RAE is present in FMS in preschool children, with those born in the first semester outperforming those born in the second semester in LM and TTS. With respect to gender, the scores obtained by girls are higher in LM and TTS.