In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling of the circadian clock plasticity and possible consequences on cytonuclear genetic make-up has not been fully elucidated. Here, we investigated the cytonuclear genetics underlying thermal plasticity of clock rhythmicity and fitness traits in reciprocal hybrid (RH) and B1K diversity panels of wild barley ( Hordeum vulgare ssp. spontaneum). Phenotypic analysis of the RH panel, showed higher abundance of plasmotype effects on chlorophyll fluorescence and its rhythmicity than plant phenology and growth. Performing a genome wide association study in the B1K panel found overlap with previously reported drivers of clock ( DOC) loci yet due to intra-chromosomal linkage disequilibrium these loci encompass shorter intervals. Moreover, by incorporating long-range chloroplastic sequencing we identified significant inter-chromosomal linkage disequilibrium and epistatic interactions between previously DOC3.2 and 5.1 loci and the chloroplastic RpoC1 genes, indicating adaptive value for specific cytonuclear gene combinations. Finally, heterologous over-expression of two barley RpoC1 alleles in Arabidopsis showed significantly differential plasticity under elevated temperatures. Our results unravel previously unknown cytonuclear interactions as well as alleles within the chloroplastic genome that control clock thermal plasticity.