An efficient Agrobacterium rhizogenes mediated transformation system of Poncirus trifoliata was developed. The combination of A. rhizogenes MSU440 with epicotyl segments was confirmed to be a suitable system for genetic transformation in P. trifoliata. About 3 weeks after inoculation with bacteria, hairy roots emerged at the cut surfaces of epicotyl segments on Murashige and Tucker selective medium free of plant growth regulators. The presence of rol B, β-glucuronidase (GUS) and yellow fluorescent protein (YFP) genes in co-transformed hairy roots was confirmed by fluorescence observation, GUS staining and polymerase chain reaction (PCR) analysis. The co-transformation rate based on GUS staining was as high as 68.3 %. A regeneration procedure of transgenic plantlets from hairy roots was also established. Out of 45 hairy root segments from different co-transformed lines, 23 were successfully regenerated into rooted plantlets. The presence and expression of YFP gene in the regenerated co-transformed plants were further confirmed by Southern blot hybridization and quantitative reverse transcription PCR. The DR5∷GUS–YFP reporter system was used to study the auxin response maxima at the apices of primary hairy roots and their lateral roots. Auxin distribution and response maxima during lateral root primordium development were also studied. The hairy roots obtained by A. rhizogenes mediated transformation were confirmed to be a good system for investigating root development in P. trifoliata. Furthermore, the genetic transformation and plant regeneration system via A. rhizogenes provides an alternative method for genetic engineering in Citrus.