Huanglongbing (HLB) is considered one of the most destructive diseases of citrus because the plants rapidly become unproductive, enter a decline, and eventually die. HLB is caused by the phloem-limited bacterium ‘Candidatus Liberibacter’ spp. The objective of this study was to evaluate seasonal variation of the in planta population of ‘Ca. Liberibacter asiaticus’ in the foliage of citrus trees in Brazil using real-time polymerase chain reaction (qPCR). Eleven plants (naturally infected, then screened) in the field with very mild and localized symptoms of HLB were confirmed to be ‘Ca. L. asiaticus’ infected by conventional PCR, and the canopies were divided into four quadrants. The bacterial population in the trees was tested on a monthly basis for up to 20 months by quantifying ‘Ca. L. asiaticus’ DNA using qPCR ‘Ca. L. asiaticus’-specific primers (As84F/As180R). The average cycle threshold (Ct) values, which relate to ‘Ca. L. asiaticus’ titer, were analyzed using a mixed model. Significant differences were observed in Ct values between seasons (F = 8.77, P = 0.0004), and abrupt changes were observed in Ct values in different quadrants of the trees. Autumn had the lowest Ct values, indicating the highest ‘Ca. L. asiaticus’ titer, and, thus, is considered the best period to detect ‘Ca. L. asiaticus’ infection in foliage of citrus trees in southern Brazil. In addition to the seasonal changes in Ct values, there was an initial decline in the Ct value in the months following initial detection, the rate of decline slowing with time. Concomitant with the increase of the bacterial population in the host, there was an increase in severity of HLB symptoms in the trees over time (Spearman’s rank correlation, r = −0.4083, P < 0.0001). The results identify the optimal season to sample foliage for ‘Ca. L. asiaticus’ in southern Brazil (autumn) and confirm the importance of sample timing to maximize detection of ‘Ca. L. asiaticus’ and, thus, contribute to the search for effective measures to manage HLB.