Purpose To study the incidence of suicide by homeless persons over a 20-year period, and identify demographic and clinical characteristics that distinguish these cases from those in non-homeless persons. Methods A comparative analysis of homeless and non-homeless persons who died by suicide between 1990 and 2009 in Queensland, Australia. Ninety-two persons (82 males and 10 females) were identified from the Queensland Suicide Register as being homeless at the time of death. Suicide rates were calculated for the second decade only due to the lack of population numbers of homeless persons in the first decade. Results Homeless persons had almost twice higher suicide rate than non-homeless counterparts. They were more often male, of young age, single/never married, non-Indigenous, unemployed, had at least one physical illness or other stressful life event prior to death, had drug and alcohol abuse problems, and also were more likely to have evidence for an untreated mental illness. Regression analysis showed that being unemployed, having a history of legal problems and not being diagnosed with mental illness were strongly associated with suicide among homeless persons. Conclusions This study is the first in Australia, and the second study internationally, to examine the characteristics of homeless people who died by suicide. Although based on a relatively small sample, the present work nonetheless carries practical implications for the development of targeted suicide prevention strategies in this peculiar population of individuals.