Abstract: Stem-end rot caused by Lasiodiplodia spp. is the most devastating disease of postharvest avocados. In order to detect the sensitivity of natural populations of Lasiodiplodia spp. to 6 systemic fungicides and their application potential in the control of avocado stem-end rot in China, 101 Lasiodiplodia spp. isolates were collected from the main avocado-producing areas in Hainan and Yunnan Provinces. The fungicide sensitivity of Lasiodiplodia spp. was determined by the mycelial growth rate method and the sensitivity baseline of the dominant species L. pseudotheobromae was established. Carbendazim, thiophanate-methyl, prochloraz, and difenoconazole exhibited strong inhibitory activities against the mycelial growth of Lasiodiplodia spp. and the mean EC₅₀ values were (0.06 ± 0.04), (0.72 ± 0.49), (0.86 ± 0.98) and (1.25 ± 1.38) μg/mL, respectively. The sensitivity frequency distribution of L. pseudotheobromae to 4 fungicides showed normal distribution with continuous, unimodal curves. Therefore, the average EC₅₀ values of mycelial growth can be used as the sensitivity baseline of Lasiodiplodia spp. There are 4% and 8% isolates with reduced sensitivity to difenoconazole and prochloraz (EC₅₀ > 5 μg/mL); 91% and 100% isolates showed very little sensitivity to pyraclostrobin and azoxystrobin with EC₅₀ values as high as (371.03 ± 353.38) and (622.86 ± 771.28) μg/mL, respectively, exhibiting non-normal distribution. Furthermore, the sequences and expression levels of the fungicide target genes in the resistance isolates were analyzed. The result showed that no mutation of CYP51 gene, the target site of prochloraz and difenoconazole, was detected based on the sequence analysis of resistance isolates. qRT-PCR analysis indicated the expression levels of CYP51 gene in resistant isolates were significantly higher than those in sensitive isolates after treatment with fungicides for 12 h, suggesting the resistance is related to overexpression of CYP51 gene. However, no point mutations or overexpression of Cyt b gene, the target of pyraclostrobin and azoxystrobin, were detected in resistant isolates. The mechanism of the resistance to pyraclostrobin and azoxystrobin still needs further exploration. Lasiodiplodia spp. were relatively sensitive to carbendazim and thiophanate-methyl and they can be used as the preferred candidate for the control of avocado stem-end rot. However, the resistance problem to the other fungicides should not be ignored.