The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of ‘Candidatus Phytoplasma’ species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by “flavescence dorée”, “bois noir” and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours. •Phytoplasmas were isolated from infected field-collected grapevine samples.•Complex media for phytoplasma isolation and growth were tested.•Unreported microaerophilic conditions are settled for phytoplasma plate colony growth.•“Flavescence dorée”, “bois noir” and aster yellows phytoplasma colonies are obtained from field infected materials.•Phytoplasma presence in colonies was detected by nested-PCR and sequencing on two genes.