*New tools were developed for the study of the endophytic development of the fungal species Fusarium equiseti and Pochonia chlamydosporia in barley (Hordeum vulgare) roots. These were applied to monitor the host colonization patterns of these potential candidates for biocontrol of root pathogens. * Molecular beacons specific for either F. equiseti or P. chlamydosporia were designed and used in real-time polymerase chain reaction (PCR) quantification of fungal populations in roots. Genetic transformation of isolates with the green fluorescent protein (GFP) gene was carried out using an Agrobacterium tumefaciens-mediated transformation protocol, and spatial patterns of root colonization were investigated by laser confocal microscopy. * Quantification of endophytes by real-time PCR in roots of barley gave similar results for all fungi, and was more accurate than culturing methods. Conversely, monitoring of root colonization by GFP-expressing transformants showed differences in the endophytic behaviours of the two species, and provided evidence of a plant response against endophyte colonization. * Both F. equiseti and P. chlamydosporia colonized barley roots endophytically, escaping attempts by the host to prevent fungal growth within root tissues. This strongly supports a balanced antagonism between the virulence of the colonizing endophyte and the plant defence response. Development of real-time PCR techniques and GFP transformants of these fungal species will facilitate future work to determine their biocontrol capacity.