AimsThe objectives of this work were to characterize molecularly the morphologically described endophyte Balansia epichloe symbiotic on three grass species, and to determine the insitu production of ergot alkaloids on these three symbiota. Methods and ResultsBalansia epichloe symbiotic with smut grass (Sporobolus poiretii), love grass (Eragrostis hirsuta) and lace grass (Eragrostis capillaries, a new host) were characterized using DNA barcoding. Laser ablation electro spray ionization (LAESI)-mass spectrometry was used to detect ergot alkaloids insitu for each symbiotum. ConclusionsThe three morphologically described symbionts on the three host grasses were indicated as belonging to the species B. epichloe, DNA barcoding suggested they were related although a cryptic species was suggested. LAESI-mass spectrometry showed that ergot alkaloids were produced invivo in two hosts but not the third although this same symbiotum was related to one of the ergot alkaloid producing symbiota as revealed by the DNA-barcoding procedure. Significance and Impact of the StudyThese results established the accumulation of ergot alkaloids in pot culture by a morpho species although there were variations with each species of grass. Barcoding described divergence among species, but considering its limitation, the suggested existence of cryptic species among this morphospecies requires substantiation by studies that are more rigorous.