The circadian system is responsible for the generation and maintenance of physiological and behavioral rhythms in mammals and allows synchronization with the environment. Different polymorphisms in clock genes have been studied in healthy humans, providing inconsistent results in different populations. In this study, we evaluated the possibility that two non-synonymous polymorphisms in PER2 (p.Gly1244Glu, rs934945) and PER3 (p.Met1028Thr, rs2640909) genes might be associated with diurnal preference in healthy Colombian subjects. A total of 209 Colombian university students were genotyped for two functional polymorphisms in PER2 and PER3 genes (rs934945 and rs2640909). We applied the composite scale of morningness (CSM) to measure phenotypic patterns of human diurnal preference. Additionally, we extracted from the CSM three subscale scores (“morningness”, “activity planning” and “morning alertness”).We used a false discovery rate approach (q values)for correction of multiple testing. PER2 (rs934945) showed a significant association with two CSM subscale scores: “activity planning” and “morning alertness”. For PER3 (rs2640909), we observed an association with the “morningness” CSM subscale scores. We found a significant association between novel and functional polymorphisms in PER2 and PER3 genes with specific CSM subscales for diurnal preference. We showed for the first time the association of rs934945 with “morning alertness” and rs2640909 with “morningness”. We suggest that these results should be replicated in order to confirm the association in other populations. Finally, the study of additional novel functional polymorphisms in other clock genes could be of relevance for a deep understanding of circadian phenotypes and neuropsychiatric disorders