The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘
Candidatus
Liberibacter asiaticus’ and ‘
Candidatus
Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘
Ca.
L. asiaticus’, which has taken over from ‘
Ca.
L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘
Candidatus
Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘
Ca.
L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘
Ca.
L. asiaticus’ was distributed throughout the inoculated plants, with mean
C
t
values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘
Ca.
L. asiaticus’ reached the highest titres in mature leaves (mean
C
t
value = 26·7) of citrus plants maintained at 22/27°C. ‘
Candidatus
Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean
C
t
value in mature leaves = 33·6).