Ficedula flycatchers are important avian speciation models. In particular, the naturally hybridizing collared flycatcher (Ficedula albicollis) and pied flycatcher (Ficedula hypoleuca) have been intensively studied in the context of speciation. Collared flycatchers and pied flycatchers are two closely related migratory passerine birds from the family Muscicapidae, which diverged approximately 1 MYA. They winter in sub-Saharan Africa and breed in woodlands across Europe, where they have several areas of sympatry and occasionally hybridize. Their F1 hybrids suffer from low to totally compromised fertility, low attractivity, and abnormal metabolic rate, which makes them a relevant model system for speciation research. A rich body of ecological information, a chromosome level genome assembly of the collared flycatcher, and protein-coding sequence, repeat sequence as well as conserved non-coding elements annotations have been established and are publicly available. Together with collaborators, we decided to extend the study system to two independent species pairs of Ficedula flycatchers, the collared flycatcher and the pied flycatcher, versus the red-breasted flycatcher (Ficedula parva) and the taiga flycatcher (Ficedula albicilla), in order to study genomic signatures of the species differentiation landscape across different evolutionary timescales. Equipped with population re-sequencing data for these four species, we perform population genomic analyses and molecular evolutionary analyses in order to better understand the role of different evolutionary processes in the build-up of the genomic differentiation landscape over time. Complementary to this macroevolutionary research angle, we also aim to expand our knowledge for our primary model system – the collared flycatcher and the pied flycatcher. For this purpose, we take advantage of novel sequencing technology and decided to generate genome assemblies of both species with help of PacBio long-read sequencing data in combination with Hi-C data, as well as to improve the genome annotation with help of PacBio Iso-Seq data. This data resource shall enable us to establish a more complete genome assembly and contribute to the identification of ‘missing genes’ in avian genomes. Moreover, the comparison of the genome assemblies among the collared flycatcher and the pied flycatcher puts us in the position to study the role of structural variants in speciation. Finally, besides evolution at the sequence level, we also investigate changes at the gene expression and epigenetic level among collared flycatchers, pied flycatchers and their F1 hybrids. This third research angle helps to improve our understanding of the functional importance of different genomic features and their interaction with each other in the hybrid genomic background. The total project thus contains of three smaller projects, ‘Speciation genomics in multiple species of Ficedula flycatchers’, ‘The role of structural variants in speciation of collared flycatchers and pied flycatchers’ and ‘Molecular mechanisms of hybrid incompatibilities among collared flycatchers and pied flycatchers’.