Methods for isolating mitochondria from different rodent tissues have been established for decades. Although the general principles for crude mitochondrial preparations are largely shared across tissues - tissue disruption followed by differential centrifugation - critical differences exist for isolation from different tissues to optimize mitochondrial yield and function. This protocol offers a unified resource for preparations of isolated mitochondria from mouse liver, kidney, heart, brain, skeletal muscle, and brown and white adipose tissue suitable for functional analysis.