Objective To screen the splicing isoforms of estrogen receptor β in the Beagle hypothalamic-pituitary-gonadal axis. Methods For ERβ mRNA CDS sequence of eight exons, primers were designed confined to the CDS sequences of two sequential exons. Beagle hypothalamus, pituitary, ovary and uterus tissue cDNA were used as template, and corresponding sequences were amplified by PCR. PCR products were sequenced and aligned in the NCBI web site. The correct gene was then analyzed with DNAMAN comparative analysis software and handwork checking up, thus got the ERβ splicing isoforms of Beagle. Results Four beagle ER beta splicing isomers were obtained: exon 4 complete skipping ER β isomer (300 bp missing), two kind of Beagle ERβ isoforms with partial exon 4 and partial exon 5 complicated missing (isoform Ⅰ334 bp missing and isoform Ⅱ 265 bp missing), and exon 7 complete missing ERβ splicing isoforms (181 bp missing). Exon 4 complete skipping and exon 7 complete missing isomers had been obtained full length coding sequence, and the other two splicing isomers were partial coding sequence. Conclusion This project gained four ER β splicing isomers of Beagle, and that will lay an important foundation for further study of their roles in the Beagle reproductive regulation mechanism.