Train and truck drivers experience a myriad of unique occupational factors, which have been postulated to contribute to a high incidence of health conditions such as depression anxiety and cardiovascular disease amongst this population. The present study aimed to identify associations between heart rate variability and negative mood states such as depression and anxiety in a cohort of Australian truck and train drivers. 120 professional drivers (60 truck drivers, 60 train drivers) were recruited from the local community. Participants complete a battery of psychometric questionnaires to assess levels of negative mood states such as depression and anxiety. Participants then completed a baseline (resting) and active (driving) task while concurrent electrocardiography data was collected to obtain heart rate variability parameters. Anxiety and depression were found to be associated with increases in low frequency heart rate variability and sympathovagal balance, and a reduction in total power. The present study identified associations between negative mood states and heart rate variability parameters that are unique to this cohort.