||A novel hemoglobin variant was observed in pure sheep (Ovis aries) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions β66(E10) and β144(HC1) of a globin chain having the sequence of the βB chain. Sequencing of the β-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG→AGG and the AAA→AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys→Arg substitution. The intrinsic oxygen affinity of the variant Hb (logP50=0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (logP50=0.92) and that of Cypriot mouflon (O. a. ophion) Hb (logP50=0.53), the latter having only the Lys→Arg change at β144, whereas nearly no differences were observed in the presence of the Cl− physiological effector. Result supports the indication that Arg at β144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at β66. Data also shows that the Lys→Arg change at β66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus. Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [βB66(E10) Lys→Arg, 144(HC1)Lys→Arg], whereas the proposed genetic nomenclature of the locus is HBBK.