Circular leaf fall disease (LFD) has been spreading in South-East Asia since 2018. This disease has been considered to be the most important factor that influences natural rubber yield. After identifying Pestalotiopsis as the causal agent of circular LFD, several studies reported the potential involvement of another primary agent. The aim of the present study was to understand the genetic basis of the infection caused by Pestalotiopsis in bioassays under controlled conditions and field observations of circular disease. Individuals from a biparental population resulting from a cross between clones PB 260 and SP 217 were phenotyped. The symptoms on leaves inoculated with two isolates of Pestalotiopsis in bioassays and natural infection in the field were found to be identical. Of the two Pestalotiopsis isolates, PE-001 produced a lesion with a larger average diameter in the population compared to PE-002. The severity of the disease observed in the field increased regularly for three years. Bioassays with Pestalotiopsis isolates and field observations revealed no correlation. The classification of genotypes has made it possible to identify classes with both small lesion diameters and low disease severity. Heritability of the lesion diameter was found to be much higher in bioassays than heritability of disease severity observed in the field. The QTLs detected differed between bioassay isolates and field observations. The latter QTLs did not remain stable over time and were no longer detected when disease severity increased in March 2023. The QTLs are associated with long chromosomal regions harbouring many genes associated with the gene ontology terms “molecular function” and “biological process”, the latter being associated with the response to biotic factors. The results of the bioassays confirmed Pestalotiopsis as the agent causing the symptoms of circular disease. This disease has a complex genetic basis which evolves over time.