The molecular methods for sex identification in birds are important resources commonly used in population studies, management and conservation plans, breeding programs and commercial poultry/farms. Recently, advanced polymerase chain reaction (PCR) methods based on melting curve analysis have been reported for Neognathae (non-ratite) birds. These genotyping strategies are simple, rapid, highly sensitive/specific and cost-effective, allowing high-throughput applicability. The evolutionary divergence and ancestral state of the sex chromosomes in the Palaeognathae (ratite) birds have hindered the utilisation of the sex-linked markers developed for non-ratite species. Therefore, the current PCR-based protocols for ratite species are restricted to agarose and polyacrylamide gels. The screening of new candidate sex-linked markers is mandatory to implement advanced PCR-based approaches in the routine molecular sexing of ratites. In this study, the nucleotide sequences of the Ostrich Z and W gametologous genes were used for the identification of new potential sex-linked markers, considering the optimisation of a molecular sexing protocol using the high-resolution melting (HRM) analysis. Four candidate markers ( NTRK2, RASEF, TMEM2 and DAPK1) were characterised for four ratite species (Ostrich, Greater Rhea, Emu and Southern Cassowary). The male and female genotypes identified were accurately differentiated based on specific melting curve profiles generated. The discussion of the polymorphic patterns obtained and their influence on the reliability of molecular sexing are complemented with an overview of the classical PCR-based methods for ratite birds. This study highlights the potential and usefulness of the recently available genomic data for ratite sex chromosomes to identify new candidate sex-linked markers with simple in silico approaches.