In view of the trend towards personalized treatment strategies for (cancer) patients, there is an increasing need to noninvasively determine individual patient characteristics. Such information enables physicians to administer to patients accurate therapy with appropriate timing. For the noninvasive visualization of disease-related features, imaging biomarkers are expected to play a crucial role. Next to the chemical development of imaging probes, this requires preclinical studies in animal tumour models. These studies provide proof-of-concept of imaging biomarkers and help determine the pharmacokinetics and target specificity of relevant imaging probes, features that provide the fundamentals for translation to the clinic. In this review we describe biological processes derived from the "hallmarks of cancer" that may serve as imaging biomarkers for diagnostic, prognostic and treatment response monitoring that are currently being studied in the preclinical setting. A number of these biomarkers are also being used for the initial preclinical assessment of new intervention strategies. Uniquely, noninvasive imaging approaches allow longitudinal assessment of changes in biological processes, providing information on the safety, pharmacokinetic profiles and target specificity of new drugs, and on the antitumour effectiveness of therapeutic interventions. Preclinical biomarker imaging can help guide translation to optimize clinical biomarker imaging and personalize (combination) therapies.