The small hepatitis B virus surface antigen (SHB) is expressed on the endoplasmic reticulum (ER) and is secreted along the cellular secretory pathway. To analyze the impact of SHB domains on its expression and secretion, SHB proteins of eight HBV genotypes (A-H) plus a clinical strain C8 were subjected to a domain analysis. All the SHBs have four transmembrane motifs (Tmhelix1-4), three regions in the ER lumen (Inside1-3), and two regions outside of the ER (Outside1-2). The topological regions of SHBs in C8 and genotypes A, B, C, D, E and G were identical, while four topological regions of SHBs in genotypes F and H were slightly different from that in C8. Deletion analysis of SHB topological regions in C8 indicated that Inside1 region was not required for SHB expression and secretion; Outside1, Tmhelix2 and Inside2 regions were required for both SHB expression and secretion; Tmhelix1 and Outside2 regions were dispensable for SHB expression but needed for SHB secretion; Tmhelix3 and Tmhelix4 regions were important for SHB expression and necessary for SHB secretion. Finer deletion (6 amino acids) mapping in Outside1 and Outside2 regions showed that amino acids 55-78 of Outside1 and Outside2 were required for SHB secretion. This is the first systematic analysis of the impact of SHB topological regions on SHB expression and secretion, which has provided valuable clues for future study of the structure-function relationship of SHB.