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The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.