The CCCTC-binding factor (CTCF) is responsible for both inter- and intra-chromosomal interactions and recruitment of essential chromatin modifiers which is vital for genome organization and function. Increasing evidence suggests a role of CTCF in sex-specific functions and sex chromosome biology. Monotremes diverged from other mammals more than 180 million years ago and evolved a complex sex chromosome system which includes multiple X and Y chromosomes and CTCF gametologues. This raises the possibility of sex-specific functions including sex chromosome-specific chromatin interactions which are observed during meiosis and mitosis for monotreme sex chromosomes. In order to understand the evolution and function of the CTCF gametologues we undertook bioinformatic, molecular cytogenetic, and chromatin immuno-precipitation sequencing (ChIP-seq) analyses in different tissues and cell lines. This revealed novel inter-chromosomal interaction between specific sex chromosomes at male interphase. Cellular localization of CTCF at meiosis revealed differences in meiotic behaviour of CTCF between monotremes and other mammals that may be related to the monotreme-specific mechanism of sex chromosome association and meiotic sex chromosome inactivation. The expression profile of CTCFX and CTCFY, and identification of the CTCF binding sites in the platypus genome support the idea that the X and Y gametologs of the CTCF protein have non-identical binding sites. Monotremes provide a unique system to explore the sex-specific functions of CTCF and our results indicate that monotreme CTCF exhibits sex-specific differences and functions and plays a role in the formation of the complex sex chromosomes system.