Aims: Formation of bacterial endospores is a basic process in Gram-positive bacteria and has implications for health, industry and the environment. Flow cytometry offers a practical alternative for the rapid detection, enumeration and characterization of bacterial endospores. Methods and Results: Paenibacillus polymyxa was chosen for this study because its spores cause sporangium deformation and have thick walls with a star-shaped section. Sporulating populations were analysed with a particle analyser and a flow cytometer after labelling with propidium. iodide and Syto-13. Flow cytometric detection of single spores was confirmed by optical and scanning electron microscopy after cell sorting. Four cell sub-populations were cytometrically detected in P. polymyxa cultures grown in liquid sporulation medium. Two sub-populations consisted of vegetative cells differing in both morphology, and viability; the other two sub-populations consisted of spores differing in their viability. Conclusions: This work has shown that flow cytometry is a simple and fast method (less than 15 minutes for sample preparation and analysis) for the study of the sporulation in P. polymyxa. The use of this technique allowed both detection and quantification of sporulation inside a culture, and distinguished cells that differed in viability despite being morphologically identical under microscopic observation. Significance and Impact of the Study: Flow cytometry has been proved to be a valuable tool for the analysis of sporulation in P. polymyxa cultures, with the unique capacity of distinguishing between endospores and vegetative cells, and between live and dead cells, in the same analysis. An important percentage of non-viable endospores has been found in aged cultures using this method.