The high carotenoid content and distinctive carotenoid profile of tritordeum are conferred by its wild progenitor, Hordeum chilense. Genetic studies on this wild barley could exploit the knowledge gained in Hordeum vulgare L. if the synteny between H. vulgare and H. chilense is established. DArTSeq markers were aligned to barley genome and used to inspect H. chilense-barley synteny. All chromosome pairs showed a good degree of collinearity with the exception of 7Hv–7Hch, where a reciprocal translocation in 7Hch was identified. Carotenoid analyses revealed a high diversity for total carotenoids, free and esterified lutein in a collection of H. chilense. Population structure analyses revealed the existence of two subgroups contrasting for total carotenoids, free lutein and esterified lutein in seeds. Lutein esters were produced with palmitic and linoleic acids as happens in tritordeum. However, tritordeum prefer palmitic acid for the synthesis of lutein esters but this preference is not maintained in H. chilense. This indicates the existence of diversity in the enzymes involved in the esterification which could be useful in tritordeum breeding. Furthermore, several accessions produced lutein monoesters but they lacked diesters which suggests that esterification is controlled by more than one enzyme in H. chilense. A total of 91 marker-trait associations were identified for carotenoid content and profile. These associations constitute a good starting point for future genetic analyses for the identification of candidate genes from H. vulgare genome.