Magnetic resonance histology (MRH) has become a widespread tool to examine brain morphology in situ or ex vivo. Samples are routinely fixed and stained to allow for longer scan times with increased contrast and resolution. Although the zebrafish is an important model for neuroscience, to date most MRH studies have focused almost exclusively on mice. In this paper, we examined, for the first time, the zebrafish brain using MRH. We compared a range of fixatives, contrast agents, and fixation/staining durations to determine optimal imaging of the zebrafish brain. By quantifying the T₁, T₂, and T₂* relaxation values, we demonstrated that ethanol and potassium permanganate are unviable for imaging and significant differences exist between mono and di‐aldehydes. Furthermore, we compared two commercially available gadolinium‐based contrast agents, Magnevist® and Optimark®, at five different concentrations. For both contrast agents, a concentration of 0.5% was determined to be ideal as it significantly shortened the T₁ but maintained a relatively long T₂ and T₂*. Subsequently, we analyzed the duration of fixation/staining and established a period of 12 h, which best minimized T₁ values but maintained T₂ and T₂* values. Finally, using this optimized fixation and staining protocol, we performed a gradient‐echo T₂*‐weighted imaging to obtain an image set of the adult zebrafish brain at an isotropic resolution of 10 µm. Copyright © 2009 John Wiley & Sons, Ltd.