Pneumocystis jirovecii pneumonia (PJP) is seen increasingly in non-human immunodeficiency virus (HIV) infected immunocompromised populations, but few cases have previously been reported in association with gemcitabine therapy. We identified all patients administered gemcitabine between March 2009 and December 2012 at the Peter MacCallum Cancer Centre. Cases of PJP were identified using accepted definitions. Overall, 288 gemcitabine-treated patients were identified. Nine cases of PJP were detected, corresponding to an overall rate of 3.1% (95% confidence interval [CI] 1.5-5.7%). PJP was diagnosed during gemcitabine therapy in seven patients, a median of 67 (range 31-109) days from commencement. Among patients with lymphoma, 4/22 developed PJP, corresponding to a rate of 18.2% (95% CI 6.1-38.2%). Fewer infections were associated with breast, lung and gastrointestinal malignancies (1/24 [4.2%], 3/118 [2.5%] and 1/61 [1.6%], respectively). A risk-based tool incorporating concomitant steroid therapy can be applied to target high-risk populations who would benefit from PJP prophylaxis during gemcitabine therapy.