AbstractEARLY FLOWERING 3(ELF3) is an important regulator of various physiological and developmental processes and hence may serve to improve plant adaptation which will be essential for future plant breeding. To expand the limited knowledge on barleyELF3in determining agronomic traits, we conducted field studies with heterogeneous inbred families (HIFs) derived from selected lines of the wild barley nested association mapping population HEB-25. During two growing seasons, phenotypes of nearly isogenic HIF sister lines, segregating for exotic and cultivated alleles at theELF3locus, were compared for 10 developmental and yield-related traits. We determine novel exoticELF3alleles and show that HIF lines, carrying the exoticELF3allele, accelerated plant development compared with the cultivatedELF3allele, depending on the genetic background. Remarkably, the most extreme effects on phenology could be attributed to one exoticELF3allele differing from the cultivated BarkeELF3allele in only one single nucleotide polymorphism (SNP). This SNP causes an amino acid substitution (W669G), which as predicted has an impact on the protein structure of ELF3. Consequently, it may affect phase separation behaviour and nano-compartment formation of ELF3 and, potentially, also its local cellular interactions causing significant trait differences between HIF sister lines.