Water availability and substrate type are essential factors in seed germination tests, influencing imbibition and seedling development. The objective of this study was to evaluate the influence of two substrates and different water volumes on the germination and vigor of chia seeds. The experimental design was completely randomized in a 2 x 6 factorial scheme, which consisted of two substrates, paper towel (Germitest®) and blotting paper, soaked with six water volumes equivalent to 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 times their dry weight, in four replicates of 50 seeds each. Germination percentage, mean time of germination, shoot length, radicle length, shoot dry matter, radicle dry matter, total dry matter (seedling), and root/shoot ratio were evaluated. Based on our results, germination tests for seeds of chia should be conducted using blotting paper (on paper) as substrate, with a water volume of 2.5 to 4.0 times its dry weight. Chia seedlings grown on blotting paper showed greater growth and dry matter accumulation compared to those grown on paper towel.