The complete cDNA sequence for walking catfish vitellogenin (VTG) (4192 bp) contained 51 bases of 5′-untranslated region, an open reading frame of 4050 bp encoding 1350 amino acids, 60 bases of 3′-untranslated region, and a poly(A) tail of 31 nucleotides. The deduced amino acid sequence of walking catfish VTG shared 58.9%, 56.9%, 41.7%, 32.6% and 8.1% identity with VTGs of common carp, zebrafish, rainbow trout, Xenopus and chicken, respectively. Results agreed with previous studies which indicated that among fishes, VTG is not a highly conserved protein, but shares a common general structure. Northern blot and RT-PCR revealed that VTG was specifically expressed in the liver of female fish. To establish the relationship between gonadosomatic index (GSI) and the level of hepatic VTGtranscript, monthly change in the GSI of females was monitored over a 1-year period from June 2006 to May 2007. The relative copy number of VTG mRNA was determined by quantitative real-time PCR. Significant variations of mean GSI values and VTG mRNA levels (P < 0.05) were observed among months. From June to August, mean GSI values ranged from 8.60 ± 0.35% to 13.07 ± 0.59%. Highest mean GSI value (18.27 ± 2.51%) and maximum VTG transcript levels were observed in September. From October through March, ovarian weight steadily declined, with the lowest mean GSI value (0.77 ±0.38%) in January. The expression profile of the VTGgene reflected the annual changes in reproductive cycle of female walking catfish, showing high levels during the breeding period and low levels during resting periods.