The zebrafish (Danio rerio) is used extensively as a model species for studies on vertebrate development and for assessing chemical effects on reproduction. Despite this, the molecular mechanisms controlling zebrafish reproduction are poorly understood. We analyzed the transcriptomic profiles of the gonads of individual zebrafish, using a 17k oligonucleotide microarray, to define the molecular basis of gender and reproductive status in sexually mature fish. The gonadal transcriptome differed substantially between genders. Among the genes over-expressed in females, eleven biological processes were over-represented including mitochondrion organization and biogenesis, and cell growth and/or maintenance. Among the genes over-expressed in males, six biological processes were over-represented including protein biosynthesis and protein metabolism. Analysis of the expression of gene families known to be involved in reproduction identified a number of genes differentially expressed between ovaries and testis including a number of sox genes and genes belonging to the insulin-like growth factor and the activin-inhibin pathways. RT-QPCR confirmed the expression profiles for nine of the most differentially expressed genes and indicated that many transcripts are likely to be switched off in one of the genders in the gonads of adult fish. Significant differences were seen between the gonad transcriptomes of individual reproductively active females reflecting their stage of maturation, whereas the testes transcriptomes were remarkably similar between individuals. In summary, we have identified molecular processes associated with gender (gonadal) specificity in breeding zebrafish and established a strong relationship between individual ovarian transcriptomes and reproductive status in females.