AIM: To evaluate the diagnostic value of B-scan ultrasound and explore the cytological characteristics of patients with vitreoretinal lymphoma (VRL) and primary central nervous system lymphoma (PCNSL). METHODS: The clinical data and pathologic specimens from patients with VRL diagnosed at the North Huashan Hospital from 2016 to 2017 were retrospectively reviewed. The patients were diagnosed by slit lamp ophthalmoscopy, B-scan ultrasound, cytology of the vitreous, which was obtained by vitrectomy, and cytokine measurements of interleukin (IL)-10 and IL-6. RESULTS: Twenty-six eyes (19.4%) out of 134 eyes of 67 patients (47 men and 20 women) with PCNSL were diagnosed with VRL by B-scan ultrasound, and 14 eyes (10.4%) were diagnosed by slit lamp ophthalmoscopy. Twenty-four eyes (17.9%) of 17 patients were confirmed as having VRL with cytology. No difference in the association between intracranial lesion location and ocular involvement was found. VRL patients had higher levels of vitreous IL-10 and IL-10/IL-6 when compared with macular hole cases, but the difference was not statistically significant. CONCLUSION: A total of 25.4% of the PCNSL patients had VRL, B-scan ultrasound examination had characteristic features and is recommended over slit lamp ophthalmoscopy for the screening diagnosis of PCNSL with intraocular involvement. Moreover, the cytological and immunohistochemical analyses performed after 25-gauge diagnostic vitrectomy were accurate diagnostic techniques.