Microbial production of chemicals has the potential to provide a sustainable source of fuels, but requires tools that can quantify and profile metabolites at the single-cell level over time. We developed longitudinal hyperspectral stimulated Raman scattering (SRS) chemical imaging to directly visualize free fatty acids in engineered Escherichia coli over many cell cycles. Compositional analysis is also developed to estimate the chain length and unsaturation of the fatty acids in living cells. This method reveals substantial heterogeneity in fatty acid production among and within colonies that emerges over the course of many generations. Interestingly, the strains display distinct types of production heterogeneity in an enzyme-dependent manner. Using fingerprint Raman signatures, we also demonstrated single-cell imaging of other valuable chemicals such as limonene and pinene, and discovered localized intracellular sites for chemical synthesis.