BackgroundPlasmodiumspecies of non-human primates (NHP) are of great interest because they can naturally infect humans.Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs ofPlasmodiuminfection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected byP. simium.MethodsWhole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting18S rRNA,Pss25andPss48/45malaria parasite transcripts. Absolute quantification was performed in positive samples for18S rRNAandPss25targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of18S rRNAandPss25transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17Pss25transcript copies per gametocyte.ResultsOverall, 87.5% of the 26 samples, previously diagnosed asP. simium,were positive for18S rRNAtranscript amplification, of which 13 samples (62%) were positive forPss25transcript amplification and 7 samples (54%) were also positive forPss48/45transcript. A strong positive correlation was identified between the Cq of the18S rRNAandPss25and between thePss25andPss48/45transcripts. The18S rRNAandPss25transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number ofPss25and18S rRNAtranscripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL.ConclusionsFor the first time, a molecular detection ofP. simiumgametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmitP. simiuminfection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-023-04601-7.