This study reassesses the transmission pattern of SARS in Singapore using viral sequence variation analysis. The authors applied mass spectrometry (MS) to detect and characterize single nucleotide variations (SNVs) in SARS-CoV isolates from different stages of the outbreak. The MS-based analysis demonstrated high sensitivity, with a 95% success rate at 75 viral RNA copies per reaction. Analysis of 13 SARS-CoV isolates identified nine SNVs that defined the molecular relationship between them, revealing a previously unidentified primary route of SARS-CoV introduction into Singapore. The study also confirmed the Singapore origin of a German patient's SARS-CoV isolate, suggesting an unnoticed aircraft transmission event. Additionally, the analysis detected heterogeneous viral sequences in primary lung tissue samples, indicating the presence of quasispecies within a single host. The authors conclude that MS-based genetic analysis is a sensitive, accurate, and cost-effective method for large-scale viral genetic characterization, particularly useful for epidemiological investigations of infectious diseases.This study reassesses the transmission pattern of SARS in Singapore using viral sequence variation analysis. The authors applied mass spectrometry (MS) to detect and characterize single nucleotide variations (SNVs) in SARS-CoV isolates from different stages of the outbreak. The MS-based analysis demonstrated high sensitivity, with a 95% success rate at 75 viral RNA copies per reaction. Analysis of 13 SARS-CoV isolates identified nine SNVs that defined the molecular relationship between them, revealing a previously unidentified primary route of SARS-CoV introduction into Singapore. The study also confirmed the Singapore origin of a German patient's SARS-CoV isolate, suggesting an unnoticed aircraft transmission event. Additionally, the analysis detected heterogeneous viral sequences in primary lung tissue samples, indicating the presence of quasispecies within a single host. The authors conclude that MS-based genetic analysis is a sensitive, accurate, and cost-effective method for large-scale viral genetic characterization, particularly useful for epidemiological investigations of infectious diseases.