The contamination of wheat by aflatoxins, mainly resulting from fungal development during storage, represents a significant public health concern due to their well-documented carcinogenic effects. This study aimed to quantify aflatoxin contamination levels (AFB1, AFB2, AFG1, and AFG2) in Triticum durum wheat samples stored in four regions of Morocco, characterised by climatic conditions ranging from semi-arid to sub-humid. Aflatoxin B1 (AFB1) was detected in 27% of the analysed samples, with quantifiable concentrations ranging from 0.025 to 20.95 & micro;g/kg. Among the AFB1-contaminated samples, 58% exceeded the regulatory limit. Traces of aflatoxin B2 (AFB2) and aflatoxin G2 (AFG2) were also detected in some samples. Aflatoxin occurrence appeared to be associated with regional climatic conditions, with higher contamination levels observed in humid coastal areas than in semi-arid regions. Molecular analysis of wheat samples corroborated the findings obtained by HPLC with fluorescence detection (HPLC-FLD) by revealing the presence of multiple Aspergillus species, including A. flavus (19.4%), A. aflatoxiformans (11.6%), A. versicolor (5.2%), A. nidulans (4.9%), and A. nomius (3.1%). Furthermore, the key genes involved in aflatoxin biosynthesis, aflR and omtA, were detected in all identified aflatoxigenic strains using multiplex PCR. Moreover, AFB1, the most toxic aflatoxin, was consistently detected in bread samples prepared from contaminated wheat grain. Although its concentration decreased by 40% to 70% during the baking process, the toxin remained present at concerning levels, underscoring the persistent health risk associated with aflatoxin contamination. These findings highlight the need for enhanced monitoring and control measures throughout the wheat production and storage chain to mitigate potential health hazards.