H2A.Z is a H2A‐type histone variant essential for many aspects of cell biology, ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues, H2A.Z.1 and H2A.Z.2, that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV, respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here, we have investigated the distinct roles of the two paralogues in cell cycle regulation and unveiled non‐redundant functions for H2A.Z.1 and H2A.Z.2 in cell division. Our findings show that H2A.Z.1 regulates the expression of cell cycle genes such as Myc and Ki‐67 and its depletion leads to a G1 arrest and cellular senescence. On the contrary, H2A.Z.2, in a transcription‐independent manner, is essential for centromere integrity and sister chromatid cohesion regulation, thus playing a key role in chromosome segregation. Synopsis This study shows that the very similar histone variants H2A.Z.1 and H2A.Z.2 have different functions in chromatin organisation and cell cycle regulation. H2A.Z.2 is essential for chromosome segregation fidelity. H2A.Z.2 regulates sister chromatid cohesion, CPC localisation and kinetochores. H2A.Z.1 is important for the G1/S transition via MYC transcription and p21/p27 suppression. H2A.Z.1 and H2A.Z.2 have distinct role in chromatin organisation and gene expression. This study shows that the very similar histone variants H2A.Z.1 and H2A.Z.2 have different functions in chromatin organisation and cell cycle regulation.