Although a rich database of Igh a allotype mouse immunoglobulin germline genes exists, current information on Igh b allotype immunoglobulin germline genes is limited. Among the immunoglobulin VH genes, single-cell amplified from six Igh b (C57BL/6 background) spleens in this study, 602 clonally independent immunoglobulin VH sequences belonging to the VH1/J558 family were identified. Whereas 335 of these sequences could be traced to have originated from 29 different VH1/J558 germline genes deposited in the NCBI Igblast database, the remaining 267 sequences appeared to have originated from 21 novel germline genes. Of the 50 VH1/J558 germline genes utilized in the peripheral repertoire of these Igh b allotype mice, the most frequently used genes included 45.21.2, V165.1, J558.6, J558.18A, and V23. Whereas the majority of the novel genes uncovered represented allelic counterparts of previously described Balb/c (Igh a allotype) genes, some appeared to represent truly novel germline genes. Collectively, the VH1/J558 germline genes exhibited high amino acid residue usage variability at the CDR1 positions, H31, H33, and H35, and the CDR2 positions, H50, H52, H53, H54, H56, and H58. The 50 VH1/J558 germline genes expressed in the peripheral Igh b repertoire also varied widely in the net charge of their CDR regions, raising the possibility that they may be differentially utilized to encode anti-nuclear autoantibodies.