Hemorrhagic fever with renal syndrome (HFRS) is a fulminant infectious disease characterized by fever, hemorrhage, renal impairment, and thrombocytopenia. Hantaviruses associated with this belong to different serotypes: Hantaan (HTN), Seoul (SEO), Dobrava/Belgrade (DOB), and Puumala (PUU). The first two, HTN and SEO, are endemic in China. To investigate the epidemiology of HFRS and virus transmission in China, we constructed prokaryotic plasmids encoding truncated recombinant HTN and SEO nucleocapsid proteins (NPs), which lacked 154 amino acid (aa), 99 aa, or 49 aa in the N-terminal region, respectively. After expression, the truncated rNPs were tested as serotyping antigens, particularly for use in the enzyme-linked immunosorbent assay (ELISA). In addition, 68 acute and 52 convalescent sera were collected from HFRS patients from Harbin, Lantian, and Kaifeng regions in China in 2004, which had hantavirus specific antibodies by IFA. A neutralization test was used to differentiate these, which showed that 73 were due to HTN infection, 33 to SEO infection, and 14 undetermined. By ELISA, the truncated rNPs, that lacked 99 (rNP100) or 49 (rNP50) N-terminal amino acids of the NPs of HTN and SEO, were able to differentiate HTNV and SEOV-specific immune sera, but the rNP155 could not. Particularly, the ELISAs based on the rNP50s had a result comparable to PRNT. Thus, the rNP50 is recommended as efficient serotyping antigen for hantavirus infection diagnosis by ELISA.