The molecular clock is a powerful technique used to estimate divergence time among organisms using molecules. Although widely used in animal and plant studies, the molecular clock is rarely applied to microbes and microbiomes: while in few cases co-radiation with host can be exploited, calibration of molecules is generally impaired by a lack of fossils and a poor knowledge of generation times outside model organisms. Here we outline, however, how molecular clocks can provide interesting insight into the biology of complex microbe-host interaction within various types of agro-ecosystems. Our case studies include: 1) the concomitant radiation of a phytoplasma with its apple host and its insect vector: a complex partnership further characterized by endosymbionts with putatively protective role against the pathogenic phytoplasma; 2) the origin of a likely beneficial new grapevine endosymbiont whose divergence matches human domestication; 3) the co-radiation of garden strawberry with its main anthracnose fungal endophytic agent. Although methodologically challenging, these examples illustrate that molecular clock is a promising and powerful tool to study the evolution of microbes and microbiomes in the agroecosystems.