The aims of the present study was to develop and describe a transvaginal ultrasound-guided biopsy method for luteal tissue in the porcine and to evaluate the effects of the method on the reproductive tract, ovarian status and pregnancy status. Biopsies were performed in four multiparous sows on Days 9 and 15 of three consecutive oestrous cycles; the size and histological composition of the samples obtained were evaluated and the reproductive tract of the sows was monitored. Furthermore, biopsies were performed in 26 multiparous sows on Days 10 and 13 after insemination, and the pregnancy rate, gestation length and subsequent litter size were evaluated. RNA was extracted from the samples obtained and the quality and quantity were determined. Altogether, 76 biopsies were performed and 38 samples were obtained. Compared with sows from which no samples were obtained (n = 6), sows from which one or more samples were obtained (n = 24) were older (parity 5.0 ± 2.8 vs 2.2 ± 0.4, mean ± s.d.), heavier (290 ± 26 vs 244 ± 27 kg) and had higher back fat (11.4 ± 2.7 vs 6.4 ± 2.5 mm; P < 0.05 for all). No effect of the biopsies (P > 0.05) was observed on the cyclicity and reproductive organs of the sows, or on corpus luteum diameter on Day 13 (8.9 ± 1.0 vs 9.2 ± 1.1 mm), pregnancy rate (95% vs 96%), gestation length (115 ± 1 vs 115 ± 1 days) and subsequent litter size (12.7 ± 2.5 vs 13.3 ± 2.8) between sows from which samples were obtained and those from which no samples were obtained. The samples obtained had a diameter of 1 mm and contained heterogeneous tissue with various cell types. The RNA quantity was 520 ± 160 µg per sample and the RNA integrity number was 8.5 ± 1.0. In conclusion, an ultrasound-guided biopsy method for ovarian tissue, which can be used for gene expression studies, was established in the porcine. No effect on corpus luteum function was found.